Diagnosis of hereditary spastic paraplegias (hsp) by detection of a mutation in the kiaa1840 gene or protein

ABSTRACT

An ex vivo method of diagnosing or predicting an hereditary spastic paraplegias (HSP) in a subject is provided which comprises detecting a mutation in the KIAA1840 gene or protein (spatacsin), wherein that mutation is indicative of an hereditary spastic paraplegias (HSP).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/567,790, filed Aug. 6, 2012, which is a continuation applicationof U.S. application Ser. No. 12/440,644, filed Jan. 4, 2010, which was a371 application of PCT/IB2007/003535, filed Sep. 11, 2007, all of saidapplications incorporated herein by reference.

The invention relates to the identification of mutations in the KIAA1840gene or protein, associated with a hereditary spastic paraplegias (HSP),and to diagnostic applications that benefit from this identification.

Hereditary spastic paraplegias (HSP) are genetically heterogeneousMendelian disorders characterized by weakness, spasticity and loss ofvibratory sense in the lower limbs (Harding et al. 1983 and Tallaksen etal. 2001). They reveal themselves clinically through difficulties inwalking possibly evolving into total paralysis of both legs. Thephysiopathology of this set of diseases is, to date, relativelyundocumented; however, anatomopathological data make it possible toconclude that the attack is limited to the pyramidal tracts responsiblefor voluntary motricity in the spinal cord (Reid, 1997). The incidenceof HSPs, which remains difficult to estimate because of rareepidemiological studies and the considerable clinical variability,varies from 0.9:100000 in Denmark, 3 to 9.6:100000 in certain regions ofSpain (Polo et al., 1991) or 14:100000 in Norway (Skre, 1974)(approximately 3:100000 in France). Various clinical and genetic formsof HSP exist. The so-called “pure” HSPs, which correspond to isolatedspasticity of the lower limbs, are clinically distinguished from the“complex” HSPs, for which the spasticity of the legs is associated withother clinical signs of neurological or non-neurological type (Bruyn etal., 1991).

Although forms of HSP have been recognized for over a century, newphenotypes are regularly described, demonstrating wide clinicalheterogeneity. Genetically, autosomal dominant (AD), autosomal recessive(AR) and X-linked inheritance are observed and almost 32 genetic locihave been identified, but only 12 genes have been cloned (Flink et al.2006). According to the putative roles of these genes, mitochondrialfunction, protein folding and axonal transport have been implicated inthe dying back of pyramidal tract axons in these disorders.

The most common forms of AD-HSP, accounting for about 40-50% of cases,are caused by mutations in the SPG4 and SPG3A genes that encode forspastin and atlastin, respectively (Hazan et al. 1990, Zhao et al. 2001and international patent application WO 01/18198). In contrast to ADforms, no major gene accounts for AR-HSP, which is less common and morevaried in clinical presentation, implying greater genetic heterogeneity.The four AR-HSP genes cloned so far, encoding for paraplegin (SPG7,MIM#607259 (OMIM database,www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM)), (Casari et al. 1998)spartin (SPG20; MIM#275900), (Patel et al. 2002) and maspardin (SPG21,MIM 248900) (Simpson et al. 2003) as well as the gene responsible forthe related spastic ataxia of Charlevoix Saguenay (ARSACS, MIM#270550)(Engert et al. 2000) probably represent less than 5% of all cases (Finket al. 2003).

A very common form of AR-HSP associates spastic paraplegia, mental orcognitive deficit and thin corpus callosum (Winner et al. 2005). Themajority of the families appear to be linked to SPG11 on chromosome 15,which was the third AR-HSP locus to be identified (Martinez et al.1999). This entity is particularly prevalent in Japan (Shibasaki et al.2000), but is also found in North-America, the Middle-East and Europe(Martinez et al 1999 and Lossos et al. 2006 and Casali et al. 2004 andWinner et al. 2004 and Stevanin et al. 2006). The typical clinicalfeatures of SPG11 consist of early-onset spastic paraplegia (usually <20years), urinary bladder dysfunction, deep sensory deficits in the legsand cognitive impairment that progress insidiously to severe functionaldisability over a period of 10-20 years. Some patients also develop arminvolvement, dysarthria, contractures and muscle atrophy. Auxiliarystudies frequently identify a thin corpus callosum (TCC) with whitematter lesions and variable cerebral cortical atrophy on magneticresonance imaging (MRI), variable cortical and thalamic glucosehypometabolism on positron emission tomography and predominantly axonalmotor or sensorimotor peripheral neuropathy on nerve conduction studies(Winner et al. 2004).

Linkage to chromosome 15q has been reported so far in 31 families inwhich the patients presented with the typical SPG11 phenotype. In theinitial study, a maximum multipoint combined LOD score of 3.14 wasdetected in seven AR-HSP families in a region between D15S1007 andD15S1012, although patients from only 2 kindreds of North-American andItalian ancestries presented with a TCC (Martinez et al. 1999). A secondstudy reported a group of 10 out of 13 Japanese families with ahomogeneous phenotype of AR-HSP-TCC with a cumulative LOD score of 9.68in the D15S971 to D15S117 interval (Shibasaki et al. 2000). Casali etal. also reported 5 Italian kindreds that showed significant linkage(Z=3.35) to the interval flanked by markers D15S1007 and D15S978 (Casaliet al. 2004). More recently, the analysis of 8 additional kindreds(Z=11.5) including 3 large consanguineous families, allowed the locus tobe restricted by the inventors to the 6 cM interval between markersD15S1044 and D15S143 (Lossos et al. 2006 and Stevanin et al. 2006) aregion that did not overlap with the interval defined in the originallymapped families (Martinez et al. 1999), therefore showing geneticheterogeneity among families linked to 15q and more closely resemblingthe locus for amyotrophic lateral sclerosis ALS5 (Hentati et al, 1998).It is of note that in the work published by Martinez et al (1999), only2 of 8 pedigrees presented with the typical SPG11 phenotype with TCC andpatients from these 2 families were linked to a larger region onchromosome 15 overlapping the region described in recent reports (Lossoset al. 2006 and Stevanin et al. 2006). More recently, the SPG11 locuswas further refined to the 4.6 cM region (according to the Marschfieldgenetic map,http://research.marshfieldclinic.org/genetics/GeneticResearch/compMaps.asp)between markers D15S968-D15S132 (Olmez et al, 2006) confirming theresults of the inventors (FIG. 2).

The inventors have now identified the gene responsible for the mostfrequent form of Autosomal Recessive Hereditary Spastic Paraplegia(AR-HSP). They have indeed demonstrated that the disease is caused bymutations in the KIAA1840 gene (also known as FLJ21439), affecting thespatacsin protein expression (Stevanin et al., 2007). This is supportedby four pieces of evidence. First, the inventors have excluded 17 out ofabout 40 genes assigned to the SPG11 candidate interval aftersignificant reduction of its size to the 3.2 cM interval (according tothe Marschfield genetic map) between markers D15S778 and D15S659 (FIGS.1 to 4). The analysis of 2 of these genes has been reported previously(Stevanin et al, 2006). Secondly, the inventors have identified 43different mutations segregating in 47 families (FIGS. 5 to 9), 16 ofwhich linked previously to the SPG11 locus with a highly significant28.1 maximal combined lod score (FIGS. 1 and 2), 8 of them alreadypublished as linked (Casali et al, 2004; Lossos et al, 2006 and Stevaninet al, 2006). Thirdly, the inventors have identified mutations, absentin at least 140 control chromosomes, that were all, leading to abnormalsplicing of the messenger RNA and/or leading to a truncated protein.Finally, the inventors have demonstrated that all mutated families,except 2 in which magnetic resonance imaging could not be performed(TUN2 and TUN14), presented with the typical AR-HSP-TCC phenotype. Inaddition, several of these families shared the same mutation withsimilar surrounding haplotypes when they came from the same geographicalorigins, suggesting regional founder effects (FIGS. 6 and 7). Mutationsin KIAA1840 affected 47 of 91 AR-HSP-TCC families in the study carriedout by the inventors making this genetic entity very frequent amongAR-HSP-TCC (75% was estimated in a previous study, Stevanin et al,2006). The invention therefore provides the identification of the majorgene responsible of AR-HSP-TCC and probably of AR-HSPs in general andopens thereby new opportunities to improve diagnosis and geneticcounseling of said disease. Moreover, the invention also provides a meanto improve the medical care management of patient affected with saiddisease. In addition, since most patients with spastic paraplegia haveisolated forms, it is conceivable that this new gene could account for asmall proportion of these patients as well. Indeed, in Europe, due tothe small size of the families, recessively inherited diseases are oftenfound in apparently isolated cases.

A first aspect of the invention thus relates to the identification ofmutations in the KIAA1840 gene or protein, associated with a hereditaryspastic paraplegias (HSP), and to diagnostic application that benefitsfrom this identification.

A second aspect of the invention relates to an isolated nucleic acid,specifically hybridizable to a region of KIAA1840 gene sequence thatcontains a mutation selected from the group consisting of

-   -   the substitutions: c.6100C>T, c.2198T>G, c. 118C>T, c. 1235C>G,        c.2833A>G, c.1951C>T, c.869+1G>A, c. 1679C>G, c.2316+1G>A,        c.2444G>T, c.2444+1G>C, c.2697G>A, c.5470C>T, c.5870C>G,        c.6091C>T, c.6477+4 A>G, c.6856C>T, c.1282A>T and c.5974C>T,    -   the deletions: c.529-533delATATT, c.6451delG, c.6832_(—)6833        delAG, c.1203delA, c.1549_(—)1550 delCT, c.6737_(—)6740 delTTGA,        c.1471_(—)1472 delCT, c.1692delA, c.2716delC, c.1668delT,        c.704_(—)705 delAT, c.5989_(—)5992 delCTGT, c.5532_(—)5533delCA,        c.5769delT, c.6739_(—)6742delGAGT, c.4307_(—)4308delAA and        c.733_(—)734delAT, and    -   the insertions: c.7029_(—)7030insT, c.2850_(—)2851insT,        c.3741_(—)3742insA, c.5982_(—)5983insCTCT, c.5986_(—)5987insT,        c.3075_(—)3076insA and c.2842_(—)2843insG.

Such an isolated nucleic acid can be used as a primer or probe.

More preferentially the invention relates to an isolated nucleic acid,which comprises a KIAA1840 gene sequence that contains one or severalmutation(s) selected from the group consisting of

-   -   the substitutions: c.6100C>T, c.2198T>G, c.118C>T, c.1235C>G,        c.2833A>G, c.1951C>T, c.869+1G>A, c. 1679 C>G, c.2316+1G>A,        c.2444G>T, c.2444+1G>C, c.2697G>A, c.5470C>T, c.5870C>G,        c.6091C>T, c.6477+4 A>G, c.6856C>T, c.1282A>T c.5974C>T,    -   the deletions: c.529-533delATATT, c.6451delG, c.6832_(—)6833        delAG, c.1203delA, c.1549_(—)1550 delCT, c.6737_(—)6740 delTTGA,        c.1471_(—)1472 delCT, c.1692delA, c.2716delC, c.1668delT,        c.704_(—)705 delAT, c.5989_(—)5992 delCTGT, c.5532_(—)5533delCA,        c.5769delT, c.6739_(—)6742delGAGT, c.4307_(—)4308delAA        c.733_(—)734delAT, and    -   the insertions: c.7029_(—)7030insT, c.2850_(—)2851insT,        c.3741_(—)3742insA, c.5982_(—)5983insCTCT, c.5986_(—)5987insT,        c.3075_(—)3076insA c.2842_(—)2843insG        or a sequence complementary thereto.

Another aspect of the invention relates to an isolated polypeptide whichcomprises the amino acid sequence of KIAA1840 containing one or severalmutation(s) selected from the group consisting of p.Q40X,p.I177_F178delfsX178, p.H235RfsX246, p.M245VfsX246, p.K401KfsX415,p.S412X, p.K428X, p.L491DfsX556, p.L517LfsX556, p.F556LfsX577, p.S560X,p.V564VfsX577, p.R651X, p.L733X, p.R815M, p.W899X, p.Q906SfsX920,p.R945G, p.R945GfsX950, p.L950FfsX953, p.V948GfsX953, p.E1026RfsX1029,p.P1248TfsX1264, p.Q1436RfsX1442, p.R1824X, p.S1844SfsX1857,p.S1923RfsX1950, p.S1957X, p.R1992X, p.L1995LfsX2000, p.C1996LfsX1999,p.L1997_(—)1998 delfsX2056, p.R2031X, p.R2034X, p.A2151PfsX2172,p.I2246_E2247delfsX2260, p.E2247_S2248delfsX2260, p.S2278LfsX2338,p.R2286X and p.V2344CfsX2349.

-   Another aspect of the invention relates to an isolated monoclonal or    polyclonal antibody that specifically recognizes a KIAA1840 protein    containing a mutation selected from the group consisting of p.Q40X,    p.I177_F178delfsX178, p.H235RfsX246, p.M245VfsX246, p.K401KfsX415,    p.S412X, p.K428X, p.L491DfsX556, p.L517LfsX556, p.F556LfsX577,    p.S560X, p.V564VfsX577, p.R651X, p.L733X, p.R815M, p.W899X,    p.Q906SfsX920, p.R945G, p.R945GfsX950, p.L950FfsX953, p.V948GfsX953,    p.E1026RfsX1029, p.P1248TfsX1264, p.Q1436RfsX1442, p.R1824X,    p.S1844SfsX1857, p.S1923RfsX1950, p.S1957X, p.R1992X,    p.L1995LfsX2000, p.C1996LfsX1999, p.L1997_(—)1998 delfsX2056,    p.R2031X, p.R2034X, p.A2151PfsX2172, p.I2246_E2247delfsX2260,    p.E2247_S2248delfsX2260, p.S2278LfsX2338, p.R2286X and    p.V2344CfsX2349.

Another aspect of the present invention relates to the use of amonoclonal or polyclonal antibody recognizing the wild type protein toidentify truncated forms of the protein.

DEFINITIONS

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon.

The term “gene” means a DNA sequence that codes for or corresponds to aparticular sequence of amino acids which comprise all or part of one ormore proteins or enzymes, and may or may not include regulatory DNAsequences, such as promoter sequences, which determine for example theconditions under which the gene is expressed. Some genes, which are notstructural genes, may be transcribed from DNA to RNA, but are nottranslated into an amino acid sequence. Other genes may function asregulators of structural genes or as regulators of DNA transcription. Inparticular, the term gene may be intended for the genomic sequenceencoding a protein, i.e. a sequence comprising regulator, promoter,intron and exon sequences.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 10, preferably at least 15, and more preferably atleast 20 nucleotides, preferably no more than 100 nucleotides, stillpreferably no more than 70 nucleotides, and which is hybridizable to aKIAA1840 genomic DNA, cDNA, or mRNA. Oligonucleotides can be labelledaccording to any technique known in the art, such as with radiolabels,fluorescent labels, enzymatic labels, sequence tags, etc. A labelledoligonucleotide may be used as a probe to detect the presence of amutated KIAA1840 nucleic acid. Alternatively, oligonucleotides (one orboth of which may be labelled) can be used for amplifying a KIAA1840nucleic acid, for instance by PCR (Saiki et al., 1988), to detect thepresence of a mutation. Generally, oligonucleotides are preparedsynthetically, preferably on a nucleic acid synthesizer. Accordingly,oligonucleotides can be prepared with non-naturally occurringphosphoester analog bonds, such as thioester bonds, etc.

A nucleic acid molecule is “hybridizable” or “hybridizes” to anothernucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when asingle stranded form of the nucleic acid molecule can anneal to theother nucleic acid molecule under the appropriate conditions oftemperature and solution ionic strength (Sambrook et al., 1989).

The conditions of temperature and ionic strength determine the“stringency” of the hybridization. For preliminary screening forhomologous nucleic acids, low stringency hybridization conditions,corresponding to a Tm (melting temperature) of 55° C., can be used,e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30 formamide,5×SSC, 0.5% SDS). Moderate stringency hybridization conditionscorrespond to a higher Tm, e.g., 40% formamide, with 5× or 6×SCC. Highstringency hybridization conditions correspond to the highest Tm, e.g.,50% formamide, 5× or 6×SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate.Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., 1989, 9.50-9.51). For hybridization with shorter nucleic acids,i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., 1989 II.7-11.8). A minimum length fora hybridizable nucleic acid is at least about 10 nucleotides, preferablyat least about 15 nucleotides, and more preferably the length is atleast about 20 nucleotides.

In a specific embodiment, the term “standard hybridization conditions”refers to a Tm of 55° C., and utilizes conditions as set forth above. Ina preferred embodiment, the Tm is 60° C. In a more preferred embodiment,the Tm is 65° C. In a specific embodiment, “high stringency” refers tohybridization and/or washing conditions at 68° C. in 0.2×SSC, at 42° C.in 50% formamide, 4×SSC, or under conditions that afford levels ofhybridization equivalent to those observed under either of these twoconditions.

As used herein, an “amplification primer” is an oligonucleotide foramplification of a target sequence by extension of the oligonucleotideafter hybridization to the target sequence or by ligation of multipleoligonucleotides which are adjacent when hybridized to the targetsequence. At least a portion of the amplification primer hybridizes tothe target. This portion is referred to as the target binding sequenceand it determines the target-specificity of the primer. In addition tothe target binding sequence, certain amplification methods requirespecialized non-target binding sequences in the amplification primer.These specialized sequences are necessary for the amplification reactionto proceed and typically serve to append the specialized sequence to thetarget. For example, the amplification primers used in StrandDisplacement Amplification (SDA) include a restriction endonucleaserecognition site 5′ to the target binding sequence (U.S. Pat. No.5,455,166 and U.S. Pat. No. 5,270,184). Nucleic Acid Based Amplification(NASBA), self-sustaining sequence replication (3SR) and transcriptionbased amplification primers require an RNA polymerase promoter linked tothe target binding sequence of the primer. Linking such specializedsequences to a target binding sequence for use in a selectedamplification reaction is routine in the art. In contrast, amplificationmethods such as PCR which do not require specialized sequences at theends of the target, generally employ amplification primers consisting ofonly target binding sequence.

As used herein, the terms “primer” and “probe” refer to the function ofthe oligonucleotide. A primer is typically extended by polymerase orligation following hybridization to the target but a probe typically isnot. A hybridized oligonucleotide may function as a probe if it is usedto capture or detect a target sequence, and the same oligonucleotide mayfunction as a primer when it is employed as a target binding sequence inan amplification primer. It will therefore be appreciated that any ofthe target binding sequences disclosed herein for amplification,detection or quantisation of KIAA1840 may be used either ashybridization probes or as target binding sequences in primers fordetection or amplification, optionally linked to a specialized sequencerequired by the selected amplification reaction or to facilitatedetection.

As used herein, the terms “KIAA1840 gene” (or its synonyms: FLJ21439,ENSG00000104133 or SPG11) denotes a KIAA1840 gene of any species,especially human, but also other mammals or vertebrates to which themethods of the invention can apply. The human KIAA1840 gene encodes alarge protein of 2443 amino-acids (aa) of unknown function that theinventors have named Spatacsin (SEQ ID NO: 2). Homo sapiens KIAA1840gene is localized on chromosome 15 and its Coding Sequence (CDS) isdeposited in Genebank under accession number NM_(—)025137, or AB058743(5′-3′ forward strand shown SEQ ID NO: 1). Human KIAA1840 gene shares85% identity with the homologous protein in dog, and 76% and 73%identity with the mouse and rat homologues and 59% with the chickenhomologue. Homology is less than 25% with orthologous proteins, ofsmaller sizes, in tetraodon and drosophila. KIAA1840 homologous proteinsat NCBI database are: dog XP_(—)544657, gallus XP_(—)413940.1, mouseBAE27954, rat XP_(—)242139.3, and at Ensembl database; drosophilaCG13531, tetraodon GSTENG00003909001. The human KIAA1840 gene contains40 exons spanning 101 Kbases of genomic DNA on chromosome 15q21.1. Theintron-exon structure of the complementary strand of the KIAA1840 geneis further indicated in Table 1 below and in FIG. 5.

TABLE 1 Exon-intron boundaries of the human KIAA 1840 gene (according to the Ensembl database)Postition in SEQ Length No Exon/Intron ID NO: 1 (bp) Sequence 5′upstream ..........cgacgcagtcaggttccggcgaaagtgaccggaagtaaccgccgggccaasequence 1 ENSE00001183257 0 258GATGGCTGCAGAGGAAGGGGTCGCGAGTGCTGCTTCCGCCGGCGGTAGCTGGGGCACCGCGGCCATGGGGCGGGTTCTACCGATGCTGTTGGTGCCAGTCCCCGCCGAGGCGATGGGGCAGCTCGGCTCCCGGGCGCAGCTGCGCACACAGCCGGAGGCTCTGGGGAGCCTGACGGCTGCGGGCAGCCTCCAAGTGCTTTCTTTGACGCCTGGCAGCCGGGGCGGGGGTCGCTGCTGCCTGGAGGGCCCCTTCTGGCA Intro 1-2 2,774gtaagtgctgagggagagttgggcc..........aataaatctaaactttttttcttag 2ENSE00001183253 258 185CTTTCTATGGGAGGATTCTCGTAACAGCAGCACACCAACTGAAAAGCCCAAACTGCTCGCTCTTGGTGAAAATTATGAACTGCTTATCTATGAATTTAATTTGAAAGATGGAAGATGTGATGCAACCATTTTGTATAGCTGTAGTAGGGAGGCATTGCAAAAGCTCATTGACGATCAAGATATCA Intro 2-3 1,128gtaagtatctacaggtggtctttca..........gaaataatatccttttgttttgtag 3ENSE00001183250 443 225GTATTTCCTTATTGTCTTTGAGAATCCTGTCATTTCACAATAACACATCATTACTGTTCATCAACAAATGTGTCATCCTACATATTATATTTCCTGAAAGAGATGCTGCAATTAGAGTACTCAACTGTTTCACACTTCCCTTGCCTGCACAGGCAGTGGACATGATTATTGACACGCAGCTCTGCAGAGGAATTCTTTTTGTTTTGAGTAGTTTAGGCTGGATCTIntro 3-4 1,782gtatccttggtggtagaagtgttga..........attttcttttaactctaactaaaag 4ENSE00001183246 668 202ACATTTTTGATGTTGTGGATGGTACATATGTAGCTCATGTGGATTTAGCACTTCACAAAGAAGACATGTGTAATGAGCAGCAACAGGAGCCAGCCAAGATTTCTTCATTTACTTCACTGAAAGTTTCTCAAGACCTCGATGTTGCAGTGATTGTCAGCTCCTCCAACTCCGCAGTTGCTCTTAACTTAAATTTGTATTTCAG Intro 4-5 4,828gtatgtagatgactgcagtttctaa..........tgtctatcattatttttaaatgtag 5ENSE00001183241 870 138GCAACACCCAGGACACCTACTGTGTGAAAGAATACTAGAAGATCTTCCTATTCAAGGACCTAAGGGCGTAGATGAAGATGATCCTGTTAACTCTGCCTACAACATGAAACTGGCCAAGTTTTCCTTCCAAATTGATAGIntro 5-6 189gtacagaaacttccttttcatgtag..........aagttatattttaccttgtttccag 6ENSE00001183238 1008 449GTCTTGGAAAGCCCAGCTATCATCATTGAATGAAACAATAAAGGAACTCCAAACTGGAGGTTTCCTGTTGTGCTCCATGGTTCCAGGATATTTTGCATTTGGAGTCACCTGAATCTGGTAACCACAGTACAAGTGTGCAGAGCTGGGCCTTCATTCCACAGGACATAATGCATGGGCAATATAATGTTCTACACGAAAGATCATGCCAAGACCAGTGATCCAGGAAGATCATGGAAAATAATGCACATCAGTGAACAAGAGGAACCCATAGAGCTTAAATGTGTGTCTGTGACAGGATTCACTGCACTGTTTACTTGGGAAGTGGAAAGGATGGGCTATACCATTACCCTCTGGGATTTGGACACCCAGCCCCATGCAGTGTTTTTCCCTTGGCACAAAGTGTATTCCTGTAGACAGTAGTGGAGACCAGCAGCTGTGCTTTGTTTTGACAG Intro 6-7 2,479gtgagactgtcttgtattagattga..........aagctaacttttatttttcctatag 7ENSE00001183236 1457 146AGAATGGACTCTCTCTGATTTTGTTTGGTTTGACTCAAGAAGAGTTTTTAAACAGACTCATGATCCATGGAAGTGCCAGCACTGTGGACACTCTTTGTCATCTCAATGGCTGGGGAAGGTGCTCAATTCCCATACATGCACTAGAGIntro 7-8 15,288gtaacagaattaaatgcccaagaac..........atttttattttcctcctcatttcag 8ENSE00001105929 1603 133GCCGGGATAGAAAATCGTCAGCTGGACACAGTAAATTTCTTTTTGAAGAGCAAGGAAAATCTTTTTAATCCATCCTCAAAATCTTCTGTATCTGATCAGTTTGATCACTTGTCATCCCATTTATATTTAAGAA Intro 8-94,116 gtaagtggaataaagatttctacat..........gttaatttctttggttctttctcag 9ENSE00001105933 1736 156ATGTGGAAGAGCTGATACCAGCATTGGATTTACTTTGCTCGGCAATTAGAGAAAGTTATTCTGAACCCCAAAGCAAACACTTTTCAGAACAATTGCTTAATCTTACACTGTCTTTCCTTAACAACCAAATAAAGGAGCTTTTCATTCACACTGAAG Intro 9-10 388gtaagaatagcagctaggaaggggg..........attggcacattggtattttccatag 10ENSE00001105923 1892 176AACTAGATGAACATCTGCAAAAAGGAGTGAACATTTTGACTAGCTACATTAATGAACTTCGAACCTTCATGATAAAGTTTCCTTGGAAGCTAACAGATGCTATAGATGAATATGATGTACATGAAAATGTCCCCAAAGTAAAGGAGAGCAATATATGGAAGAAACTCAGCTTTGAG Intro 10-11 2,161gtaagtacgaataatcatcacttct..........aaggcaaacgtttttcttttcctag 11ENSE00001105941 2068 177GAAGTTATTGCCAGCGCCATTTTAAACAACAAAATACCAGAGGCACAGACTTTCTTCAGGATTGATAGTCATTCTGCTCAAAAACTTGAGGAGCTTATTGGCATAGGCCTAAATTTGGTCTTTGACAATTTAAAAAAGAACAATATAAAGGAAGCCTCTGAACTTTTGAAGAATATG Intro 11-12 3,531gtgagtggtgtaatccataaagtct..........ttttggttttctatgtttattttag 12ENSE00001183220 2245 72GGGTTTGATGTAAAAGGCCAATTGCTCAAGATCTGCTTCTATACAACTAATAAAAATATACGTGACTTTTTGIntro 12-13 380gtaggtaaaggtgagactacatagt..........ctgctttaattactttttattcaag 13ENSE00001183213 2317 128GTTGAAATTTTAAAAGAAAAAAATTATTTTTCTGAAAAAGAGAAAAGAACTATAGACTTCGTGCATCAAGTTGAGAAGCTTTATTTGGGACATTTCCAAGAAAATATGCAAATCCAGTCATTTCCCAG Intro 13-14 285gtagtctcattagtcctcttttgat..........aaaaaatttatatcactgtttttag 14ENSE00001183208 2445 176GTACTGGATAAAGGAACAAGATTTTTTCAAGCACAAGTCTGTTTTGGACTCATTCCTGAAATATGATTGTAAAGATGAATTTAACAAACAGGACCATAGAATTGTGTTAAATTGGGCTCTGTGGTGGGATCAACTAACACAAGAATCCATCCTTCTCCCCAGGATAAGTCCAGAAG Intro 14-15 1,355gcaagtgtgagagagcctgaaatat..........ttaaaatgtgttttttttcatgtag 15ENSE00001183204 2621 214AATACAAATCATATTCCCCTGAAGCCCTCTGGAGATACCTCACAGCTCGCCATGATTGGTTAAACATTATCTTATGGATTGGAGAATTTCAAACCCAGCATAGTTATGCTTCACTTCAGCAGAACAAATGGCCCCTTCTGACTGTTGATGTTATTAACCAGAATACTTCCTGTAACAACTACATGAGGAATGAAATTTTAGATAAGCTGGCCAGIntro 15-16 4,623gtattataactgttgaactaatacc..........tgacatcctataaatctgtccatag 16ENSE00001047610 2835 204GAATGGGGTTTTTTTGGCATCTGAACTGGAAGACTTTGAATGCTTCCTCCTAAGACTGAGCCGTATTGGAGGTGTAATACAGGATACCCTCCCTGTTCAAAACTACAAGACCAAAGAAGGTTGGGATTTCCATTCTCAATTCATTCTCTATTGTTTGGAGCACAGTCTGCAGCATCTTCTTTATGTCTACCTTGACTGTTACAA Intro 16-171,826 gtgagtactgagaatgcatttgtcc..........aggtttttgtttgttttatatacag 17ENSE00001287244 3039 107ACTTAGTCCTGAAAATTGTCCCTTTTTGGAAAAAAAAGAGTTACATGAAGCACACCCTTGGTTTGAATTTTTAGTTCAGTGTCGACAAGTTGCCAGTAACTTAACAG Intro 17-18 2,444gtatgggtatactgtattaaacaca..........aaaaacactgtcttttattttccag 18ENSE00001047605 3146 146ATCCCAAACTGATCTTCCAGGCTAGCCTTGCAAATGCTCAGATTTTGATTCCCACCAATCAGGCCAGTGTAAGCAGTATGCTATTGGAAGGACATACCCTCCTGGCCCTTGCTACTACAATGTATTCTCCTGGGGGTGTCAGTCAGIntro 18-19 2,234gtatggatagcactttatgacaaaa..........acctgttatctgtttttttacttag 19ENSE00001047617 3292 162GTTGTTCAGAATGAAGAAAATGAAAACTGTTTGAAGAAAGTGGATCCCCAGCTATTGAAGATGGCATTAACTCCTTACCCCAAGCTAAAAACTGCTCTCTTCCCACAGTGCACTCCTCCTAGTGTCCTGCCATCTGATATTACAATCTACCACCTTATTCAG Intro 19-20 2,352gtacagtatttaggtggccaatatt..........ctgtttaacttttcccctttttcag 20ENSE00001047612 3454 67TCATTATCACCCTTTGATCCTAGCAGATTGTTTGGCTGGCAGTCTGCTAACACACTAGCTATAGGAGIntro 20-21 5,392gtaagtcatcatgggtacttcttga..........taatattgttttactttccccctag 21ENSE00001047594 3521 166ATGCATGGAGTCATCTCCCACATTTCTCTAGCCCTGACCTGGTTAATAAATATGCTATAGTGGAACGTCTGAATTTTGCTTATTATTTACATAATGGGCGGCCATCATTTGCATTTGGTACTTTTCTGGTCCAGGAATTAATCAAGAGCAAGACTCCCAAGCAGCT Intro 21-22 1,630gtgagtatttaaaatataattttgt..........tgattttgattcctttctttttcag 22ENSE00001047598 3687 206GATCCAGCAAGTAGGCAATGAAGCCTATGTTATAGGGCTCTCCTCCTTCCACATACCTTCAATAGGAGCTGCATGTGTTTGTTTCTTAGAATTGCTTGGCCTTGACAGCCTCAAGCTCAGAGTTGATATGAAAGTGGCCAATATAATTTTGAGCTACAAGTGCAGAAATGAAGATGCTCAGTACAGCTTTATCAGAGAGTCTGTAG Intro 22-23257 gtacagcaccttttatctggcctgc..........attttgttgtttatatttcttacag 23ENSE00001047622 3893 109CCGAAAAACTATCTAAACTAGCTGATGGTGAAAAGACAACCACAGAAGAATTGCTTGTTCTCTTAGAAGAAGGTACATGGAACAGCATTCAGCAACAGGAAATAAAGAG Intro 23-24 1,321gtttgtgagttgcagtctcagcctc..........cccccacctctaattctgattatag 24ENSE00001047619 4002 160GTTATCCAGTGAATCTAGCAGCCAATGGGGCATTAGTGGTGCAGTTCTGCAGGCTACACAATATGAAACTAAGCATATCTTACCTTAGAGAATGTGCCAAAGCAAATGATTGGCTGCAGTTCATTATTCACAGCCAACTCCACAACTACCACCCAGCAGAG Intro 24-25 428gtaagccactaattgttagcagtca..........tttaatcatctgatatgccttctag 25ENSE00001047603 4162 273GTGAAATCCCTTATCCAGTACTTCAGCCCAGTCATTCAAGACCACTTAAGGCTGGCTTTTGAGAACTTGCCCTCAGTGCCCACCTCCAAAATGGACAGCGATCAAGTCTGCAATAAGTGCCCCCAGGAACTTCAAGGAAGCAAACAAGAGATGACCGATTTATTTGAAATTCTGCTCCAATGCTCAGAGGAGCCAGACTCCTGGCACTGGCTTCTGGTTGAAGCAGTGAAACAACAGGCCCCTATCCTCAGTGTTCTGGCCTCATGTCTCCAG Intro 25-26 623gtgaggatcatgagaagcctgaagt..........tgttattttatttatcccgtggcag 26ENSE00001047590 4435 201GGTGCCAGTGCCATTTCTTGTCTCTGTGTTTGGATCATCACTTCTGTGGAGGACAATGTTGCAACTGAAGCAATGGGACACATTCAGGACTCAACAGAGGACCATACCTGGAACCTTGAGGATCTTTCAGTCATCTGGAGAACATTATTAACAAGACAAAAGAGCAAAACTCTCATCAGAGGTTTCCAGCTTTTCTTTAAG Intro 26-27 2,820gtagtgatagttgcttcacttcttt..........atttttttcaaactctttgtcaaag 27ENSE00001047613 4636 108GATTCCCCGTTACTACTGGTGATGGAGATGTATGAACTGTGTATGTTCTTCAGGAATTATAAAGAAGCTGAAGCTAAACTTCTGGAGTTTCAGAAGAGCCTTGAAACG Intro 27-28 2,916gtaagttggaattatggtgctcttt..........ctaagcttctctttttctttcatag 28ENSE00001047595 4744 163CTTAACACAGCAGCCACAAAGGTCCACCCTGTCATCCCTGCCATGTGGCTGGAGGATCAGGTGTGTTTCCTTTTGAAGCTTATGCTACAGCAGTGTAAGACCCAGTATGAGCTGGGGAAGCTTTTACAGCTCTTTGTTGAAAGAGAGCATCTCTTCTCTGATG Intro 28-29 3,401gtaagacaatccttacagttaagtt..........ttatatccttttctctttggcacag 29ENSE00001047608 4907 215GTCCAGATGTGAAAAAGCTTTGCATCCTTTGCCAGATTTTGAAGGATACATCCATAGCCATTAATCATACAATTATTACCAGCTACAGCATTGAGAATCTTCAGCATGAATGTGATCTATTTTGGAAAGACTGCAGACAGATGGACAATTCGCTTTGGCCAGGAGGGTAGCAGAATTAGCTGAGTTACCTGTGGACAACTTGGTTATTAAAGAGIntro 29-30 1,077gtatcatcggtcttttttttttttt..........aaatctgctttgttaaatttcacag 30ENSE00001047607 5122 745ATAACACAGGAAATGCAGACCCTAAAACACATTGAACAGTGGTCACTAAAACAAGCAAGAATTGACTTCTGGAAAAAATGCCATGAGAATTTTAAGAAAAATTCAATTTCAAGCAAAGCAGCTTCTTCCTTTTTCTCAACCCAGGCCCATGTGGCATGTGAGCACCCAACTGGATGGAGCAGCATGGAGGAGCGCCATCTGCTGCTCACCTTGGCAGGGCACTGGCTTGCCCAGGAGGACGTGGTGCCCTTGGATAAGCTGGAGGAGCTGGAGAAGCAGATCTGGCTGTGCCGCATCACCCAGCACACTCTTGGAAGAAATCAGGAGGAAACAGAGCCCAGATTTTCTCGACAGATCTCAACTAGTGGTGAACTTTCCTTTGATAGTTTAGCCAGTGAGTTTTCCTTCTCCAAGTTGGCTGCTCTGAACACATCAAAATACTTAGAACTTAACAGCCTTCCATCCAAAGAGACATGCGAGAATAGATTGGATTGGAAAGAGCAGGAGTCACTAAACTTTTTGATTGGGCGCCTACTGGATGATGGCTGTGTGCATGAAGCAAGTAGAGTATGCCGGTATTTTCATTTTTATAATCCAGATGTCGCCTTGGTATTGCACTGCAGAGCACTGGCCTCAGGGGAAGCTAGTATGGAGGATCTGCACCCAGAGATCCATGCTCTCCTACAAAGTGCTGAGCTGCTTGAGGAAGAAGCACCCGACATTCCCCTAAGGAGAGTCCACAGCA Intro 30-31 8,772gtaagtgaaggagatcagatggccc..........ccctcagacttgtatttgcttccag 31ENSE00001047614 5867 140CTTCAAGTCTGGATAGTCAGAAGTTTGTGACAGTGCCCTCCAGTAATGAAGTGGTAACTAACCTGGAAGTGCTGACAAGCAAATGCCTCCATGGGAAGAACTACTGTCGACAGGTCCTCTGTCTGTATGATCTTGCCAAGIntro 31-32 1,156gtatgtgccaaggggtggggctcct..........ttgactggctttgtcttcctctcag 32ENSE00001123435 6007 199GAGTTGGGCTGTTCCTACACAGATGTTGCTGCTCAGGATGGTGAAGCCATGCTCCGGAAAATCTTGGCCTCTCAGCAGCCTGACCGATGCAAACGAGCCCAGGCCTTCATCAGCACACAGGGCCTTAAGCCAGATACTGTGGCTGAACTCGTGGCAGAAGAGGTGACACGGGAGCTGCTTACTTCATCACAGGGAACAG Intro 32-33 726gtgccctaccccccggggattccca..........cctgtcttcacacctctctgtacag 33ENSE00001123426 6206 138GACATAAGCAGATGTTCAACCCAACAGAGGAAAGCCAGACATTTCTTCAGCTGACCACTCTGTGTCAAGACCGCACATTGGTAGGCATGAAGTTGTTGGATAAGATTTCCTCCGTTCCCCATGGGGAACTGTCTTGCAIntro 33-34 2,024gtaagttattgacctttttcttaca..........atcttaccagtgcccaccctcccag 34ENSE00001123415 6344 134CCACAGAGCTCCTGATCCTGGCCCATCATTGCTTCACCCTGACGTGCCACATGGAGGGCATCATCCGAGTCCTACAGGCCGCCCACATGCTCACAGATAACCACCTGGCCCCCAGTGAGGAGTATGGGCTGGTGIntro 34-35 1,019gtaagtagccccctcaaccccagtc..........tgcgagctgtcctcccacttcacag 35ENSE00001123405 6478 108GTACGGCTCCTCACTGGCATTGGAAGGTACAACGAGATGACATACATATTTGATTTGCTGCATAAAAAGCACTACTTTGAAGTGCTAATGAGGAAGAAGTTGGATCCG Intro 35-36 1,805gtaggtgcaaagtaatgagctccag..........gctttttcccttttattctgggcag 36ENSE00001123397 6586 169AGTGGTACCCTGAAAACAGCCCTGCTGGACTACATCAAACGCTGCCGTCCTGGAGACAGTGAAAAGCACAATATGATTGCCCTGTGCTTCAGCATGTGCCGGGAGATTGGCGAGAACCACGAGGCAGCTGCCCGCATCCAACTGAAATTGATTGAGTCTCAGCCCTGGG Intro 36-37 1,118gtgagtgaggtcacagccacactac..........caaatcttctttatttcccctacag 37ENSE0000684756 6755 89AGGACAGCCTCAAGGATGGGCACCAGCTGAAACAACTGCTGCTGAAGGCCCTGACTCTGATGTTGGATGCAGCAGAGAGTTATGCCAAG Intro 37-38 207gtaacccaaaggcttttttcagact..........gtgcctctccacccttgttcctcag 38ENSE0000684735 6844 156GACTCCTGTGTGCGACAGGCCCAGCACTGTCAGCGGCTCACCAAGTTGATAACTCTGCAGATTCACTTTCTGAACACTGGCCAGAACACAATGCTCATCAACTTGGGCCGCCACAAGCTGATGGACTGTATTCTGGCCCTACCTCGGTTCTACCAG Intro 38-39 1,155gtgagcaagaaagcaaactgtagcc..........gtccttcttcacctctccttttaag 39ENSE0000684706 7000 152GCTTCTATTGTGGCTGAGGCCTACGATTTTGTTCCAGATTGGGCTGAAATTTTATACCAGCAAGTGATTCTTAAAGGAGACTTTAATTACTTGGAAGAATTTAAGCAGCAAAGGTTATTAAAGTCCAGTATATTTGAAGAGATTTCCAAAAA Intro 39-40 1,245gtaagtattaaaagttgactgtaaa..........ctgtacattatgtttctttatctag 40ENSE0000884381 7152 600ATATAAACAACATCAGCCTACTGACATGGTCATGGAAAACCTGAAGAAATTACTCACATATTGTGAAGATGTTTACCTGTATTACAAGTTGGCATACGAACACAAGTTTTATGAAATTGTAAATGTGCTTCTGAAGGACCCTCAGACAGGTTGCTGTCTAAAGGACATGCTAGCAGGTTAGATGATTTCATAGGTGTCTGTTTTCTTGTACTGTTAGCAGATTCTGACAGATGTGATGAGAAGAAGAATGCATTGGAGATCTTTGCTAAAGTTGAACAATCCCGGTACTGTACCATATCAGTCCTTTGTGGGTAGTAGGTAGCAAGTAAGAAACTTTTCAGGAGGAAATTCCTATTTAAAATAGATTGATTTTAGATGATTGTTCATCCACACCATTTTATATAGATACTAGTATTAAGATCAAAAGCTTCCTCTTCCTCAGGACAGCTTCTACTTTAGATGATCCAATAATGATTAAAGAATACCTGTACCTGCAGATTCCAGTTTCAAAGAAATTTAATTATTATTTACACAGTTAAGGAACAGGTGATACATTTTCATTTGTTAGAAACTGATCTTTCTGTAATAAAATAGATTTTC 3′ downstreamaattcagtgtatgtcattattactgctaaggaaatcttagcccttgtctg.......... sequence

As used herein, the term “Spatacsin” denotes the SPAsticity with Thin orCorpus callosum Syndrom protein, which is encoded by the KIAA1840 gene.The sequence of the human form is shown in SEQ ID NO:2.

The terms “mutant” and “mutation” mean any detectable change in geneticmaterial, e.g. DNA, RNA, cDNA, or any process, mechanism, or result ofsuch a change. This includes gene mutations, in which the structure(e.g. DNA sequence) of a gene is altered, any gene or DNA arising fromany mutation process, and any expression product (e.g. protein orenzyme) expressed by a modified gene or DNA sequence. Generally amutation is identified in a subject by comparing the sequence of anucleic acid or polypeptide expressed by said subject with thecorresponding nucleic acid or polypeptide expressed in a controlpopulation. A mutation in the genetic material may also be “silent”,i.e. the mutation does not result in an alteration of the amino acidsequence of the expression product.

In the context of the instant application, mutations identified inKIAA1840 gene are designated pursuant to the nomenclature of Den Dunnenet al. 2001 (http://www.genomic.unimelb.edu.au/mdi/mutnomen/). Asdefined by Dunnen and Antonarakis at the nucleic acid level,substitutions are designated by “c. position(nt)>(nt)”, e.g. “c.118C>Gdenotes that at nucleotide 118 of the reference sequence C is changed toa G. The mutation at the protein level is denoted p.Q40X: which meansthat a glutamine (Q) at position 40 encoded by CAG is replaced by a STOP(TAG) codon (Q40X). Deletions are designated by “del” after the deletedinterval (followed by the deleted nucleotides). For instance529_(—)533delATATT denotes a ATATT deletion from nucleotides 529 to 533.The consequence of this deletion, p.I177_F178delfsX, is a deletion ofaminoacids at positions 177 and 178 and a frameshift (fs) in the codingsequence leading to the appearance of a premature STOP codon (X). Analternative nomenclature is to indicate the position of the stop codonin the resulting protein after the X; p.I177_(—)178 delfsX178 indicatesthat the stop codon resulting from the mutation is at codon 178.Insertions are designated by “ins,” followed by the insertednucleotides. For example, c.7029_(—)7030 insT denotes that a T wasinserted after nucleotide 7029. This leads to the replacement of valine(V) by cysteine (C) at position 2344 and to a frameshift of the codingsequence and a premature STOP codon at amino-acid 2349 (fsX):p.V2344CfsX or p.V2344CfsX2349. When a mutation is predicted to alterthe splicing of the mRNA because the variant modifies a nucleotide ofthe consensus sequence for splicing (acceptor or donor site), the “r.?”denotes that the consequences of the mutation could not be checked atthe RNA level, but is likely (as verified athttp://rulai.cshl.edu/new_alt_exon_db2/HTML/score.html).

The term “hereditary spastic paraplegias (HSP)” denotes geneticallyheterogeneous Mendelian disorders characterized by weakness, spasticityand loss of vibratory sense in the lower limbs. The term “AutosomalRecessive Hereditary Spastic Paraplegia” or “AR-HSP” denotes spasticparaplegia that is transmitted as an autosomal recessive trait. Patientswith HSP or AR-HSP can have a pure phenotype, or, more often, a complexphenotype that associates various neurological signs (cerebellar ataxia,mental retardation, peripheral neuropathy, etc). The term “AR-HSP-TCC”denotes an AR-HSP with Thin Corpus Callosum usually associated with,mental or cognitive deficit and peripheral neuropathy. Families withoutproved TCC can also be mutated in this gene either because of slowprogression of the disease in the patient or because magnetic resonanceimaging (MRI) couldn't be performed due to patient refusal orimpossibility (patients leaving far from cities in North-Africa—this isthe case for families FSP400, FSP393 and FSP343).

As used herein, the term “subject” denotes a mammal, such as a rodent, afeline, a canine, and a primate. Preferably a subject according to theinvention is a human.

Mutations in the KIAA1840 Gene and Spatacsin Protein

The inventors identified various mutations in the KIAA1840 gene.

Fortythree different mutations on human KIAA1840 gene were indeedidentified in 47 families, including the 16 linked ones, all at thehomozygous state, except in 16 kindreds. They were either nonsensemutations (n=13), deletions (n=17), insertions (n=7), or splice sitemutations (n=6) in the coding sequence, and resulted theoretically in anabnormally spliced mRNA or a truncated protein in all cases.

In one family, linked with a maximal 3.1 multipoint lod score to SPG11,a missense R945G mutation segregated at the homozygous state in bothpatients and was not detected in 150 control chromosomes. The mutationis probably not only affecting the nature of the amino-acid. Position ofthis variant was in the 5′-splice site consensus sequence (2 basesbefore the end of exon 15). The score of the 5′-splicing sequencechanged from 4.9 for the wild type to 2.7 for the variant (AlternativeSplicing Database::http://rulai.cshl.edu/new_at_exon_db2/HTML/score.html) suggesting thatthis variant could act at both the RNA level (splicing effect) and atthe protein level (missense change). Indeed, this was confirmed bydirect sequencing (using primers GCTCTGTGGTGGGATCAACT andTGCTTACACTGGCCTGATTG) on mRNA isolated from lymphoblasts of an affectedfamily member (FSP670-5) in which an alternative splice site isgenerated downstream in intron 15 leading to a 65 bp insertion and apremature stop codon (c.2833A>G, r.2834+1_(—)2834+65 ins,p.R945GfsX950). It cannot be excluded, however, that splicing occurs atits normal place in a small amount of messenger RNA and that a fulllength protein is generated with the G variant at position 945.Similarly, the mutation c.2444G>T, p.R815M likely affects not only theamino-acid but also splicing of exon 13 since the splice score down from3.7 to 0.2 for the mutation. In addition, the c.869+1G>A, c.2316+1G>A,c.2444+1G>C and c.6477+4A>G, are all clearly affecting the acceptorsplicing consensus sequence (see splice scores in table 2) and likelyalter the splicing of exons 4, 12, 13 and 34, respectively. Themutations identified by the inventors are presented on the followingTable 2.

TABLE 2 Mutations identified in the KIAA1840 CDS (SEQ ID NO: 1) andprotein (SEQ ID NO:2) Mutations Family code Family code Ex- mRNA orProtein SEQ (Origins) at the (Origins) at the ons Nucleotide variantsvariants ID NO: homozygous state heterozygous state 1 c.118C>T, p.Q40X149 FSP672 (Israel) 3 c.529_533delATATT p.I177_F178>S177fsX1 150 FSP386(Portugal) ITA17 (Brazil) 78 FSP754 (Portugal) FSP831 (Portugal) 4c.704_705delAT p.H235RfsX246 166 SPD199 (Turkey) 4 c.733_734delAT,p.M245VfsX246 151 TUN2 (Tunisia) FSP117 (France) TUN3 (Tunisia) TUN4(Tunisia) TUN22 (Tunisia) In- c.869+1G>A r.? FSP847 (Argentina) tron 4Splice score down from 9.8 to −0.9 6 c.1203delA, p.K401KfsX415 152 PE(Italy) 6 cv.1235C>G, p.S412X 153 FSP393 (Portugal) 6 c.1282A>T p.K428X167 FSP830 (Portugal) 7 c.1471_1472delCT p.L491DfsX556 168 FSP522(France) 7 c.1549_1550delCT, p.L517LfsX556 154 FSP343 (Algeria) (nontypical) 8 c.1668delT p.F556LfsX577 169 SAL646 (France) 8 c.1679C>Gp.S560X 170 ITA16SB (Italy) 8 c.1692delA p.V564VfsX577 171 DKD (Italy)10 c.1951C>T, p.R651X 155 MP (Italy) FSP683 (Romania) ITA28VAC (Italy)11 c.2198T>G, p.L733X 156 OS (Italy) In- c.2316+1G>A r.? FSP892 (Norway)tron 12 Splice score down from 6.2 to −4.5 13 c.2444G>T p.R815M and/orr.? 172 ITA28VAC (Italy) Splice score down from 3.7 to 0.2 In-c.2444+1G>C r.? ITA16 (Brazil) tron 13 Splice score down from 3.7 to −715 c.2697G>A p.W899X 173 ITA10 (Italy) 15 c.2716delC p.Q906SfsX920 174ITA9 (Italy) 15 c.2833A>G, r.2834+1_2834+65ins, 165 FSP670 (Israel)ITA14 (Italy) p.R945G or 188 p.R945GfsX950 Splice score down from 4.9 to2.7 16 c.2842_2843insG, p.V948GfsX953 157 PE (Italy) 16 c.2850_2851insT,p.L950FfsX953 158 MP (Italy) 17 c.3075_3076insA p.E1026RfsX1029 175 ITA8(Germany) 22 c.3741_3742insA p.P1248TfsX1264 176 ITA17 (Brazil) 25c.4307_4308delAA p.Q1436RfsX1442 177 FSP398 (Israel) ITA16 (Brazil) 30c.5470C>T p.R1824X 178 ITA8 (Germany) 30 c.5532_5533delCAp.S1844SfsX1857 179 FSP522 (France) 30 c.5769delT p.S1923RfsX1950 180FSP838(Saudi-Arabia) 31 c.5870C>G p.S1957X 181 ITA16SB (Italy) 31c.5974C>T, p.R1992X 159 FSP117 (France) 31 c.5982_5983insCTCTp.L1995LfsX2000 182 DKD (Italy) 31 c.5986_5987insT p.C1996LfsX1999 183FSP398 (Israel) 31 c.5989_5992delCTGT p.L1997_Y1998>M1997f 184 FSP683(Romania) sX2056 32 c.6091C>T p.R2031X 185 ITA1 (Turkey) 32 c.6100C>T,p.R2034X 160 FSP446 (Morocco), FSP221 (Algeria), FSP732 (Algeria),FSP400 (Algeria) FSP792 (Algeria) FSP845 (Morocco) TUN9 (Tunisia) TUN12(Tunisia) TUN14 (Tunisia) 34 c.6451delG, p. A2151 P fsX2172 161 SAL1608(France) In- c.6477+4 A>G r.? FSP830 (Portugal) tron 34 Splice scoredown from 9.6 to 6.6 36 c.6737_6740delTTGA, p.I2246_E2247>S2246f 162FSP920 (Japan) FSP343 (Algeria) sX2260 36 c.6739_6742delGAGTp.E2247_S2248>L2247f 186 SAL646 (France) sX2260 37 c.6832_6833delAG,p.S2278LfsX2338 163 FSP75 (Portugal) 38 c.6856C>T p.R2286X 187 ITA14(Italy) 39 c.7029_7030insT, p.V2344CfsX2349 164 FSP515 (Tunisia)

Each mutation are herein numbered according to human KIAA1840 CDS andamino acid sequence as shown in SEQ ID NO: 1 and SEQ ID NO:2.

Accordingly, the invention relates to an isolated nucleic acidspecifically hybridizable to a region of KIAA1840 gene coding sequence(SEQ ID NO:1) that contains a mutation selected from the groupconsisting of

-   -   the substitutions c.6100C>T, c.2198T>G, c.118C>T, c.1235C>G,        c.2833A>G, c.1951C>T, c.869+1G>A, c. 1679 C>G, c.2316+1G>A,        c.2444G>T, c.2444+1G>C, c.2697G>A, c.5470C>T, c.5870C>G,        c.6091C>T, c.6477+4 A>G, c.6856C>T, c.1282A>T and c.5974C>T,    -   the deletions: c.529-533delATATT, c.6451delG, c.6832_(—)6833        delAG, c.1203delA, c.1549_(—)1550 delCT, c.6737_(—)6740 delTTGA,        c.1471_(—)1472 delCT, c.1692delA, c.2716delC, c.1668delT,        c.704_(—)705delAT, c. 5989_(—)5992delCTGT, c. 5532_(—)5533delCA,        c.5769delT, c.6739_(—)6742delGAGT, c.4307_(—)4308 delAA and c.        733_(—)734delAT, and    -   the insertions: c.7029_(—)7030insT, c.2850_(—)2851insT,        c.3741_(—)3742insA, c. 5982_(—)5983insCTCT, c.        5986_(—)5987insT, c. 3075_(—)3076insA and c.2842_(—)2843insG.

In one embodiment of this aspect of the invention, the isolated nucleicacid according to the invention consists of at least 10 nucleotides,preferably 20 nucleotides, more preferably 40 nucleotides.

In a preferred embodiment, such an isolated nucleic acid is specificallyhybridizable to a region consisting of 10 nucleotides upstream and 10nucleotides downstream of a mutation selected from the group consistingof

-   -   the substitutions: c.6100C>T, c.2198T>G, c.118C>T, c.1235C>G,        c.2833A>G, c.1951C>T, c.869+1G>A, c. 1679 C>G, c.2316+1G>A,        c.2444G>T, c.2444+1G>C, c.2697G>A, c.5470C>T, c.5870C>G,        c.6091C>T, c.6477+4 A>G, c.6856C>T, c.1282A>T c.5974C>T,    -   the deletions: c.529-533delATATT, c.6451delG, c.6832_(—)6833        delAG, c.1203delA, c.1549_(—)1550 delCT, c.6737_(—)6740 delTTGA,        c.1471_(—)1472 delCT, c.1692delA, c.2716delC, c.1668delT,        c.704_(—)705delAT, c. 5989_(—)5992delCTGT, c. 5532_(—)5533delCA,        c.5769delT, c.6739_(—)6742delGAGT, c.4307_(—)4308 delAA c.        733_(—)734delAT, and    -   the insertions: c.7029_(—)7030insT, c.2850_(—)2851insT,        c.3741_(—)3742insA, c. 5982_(—)5983insCTCT, c.        5986_(—)5987insT, c. 3075_(—)3076insA c.2842_(—)2843insG, of the        KIAA1840 gene sequence.

Preferably, “specifically hybridizable” means “hybridizable underconditions at 68° C. in 0.2×SSC, at 42° C. in 50% formamide, 4×SSC, orunder conditions that afford levels of hybridization equivalent to thoseobserved under either of these two conditions”.

In an alternative manner, a sequence “specifically hybridizable” to atarget sequence means a sequence showing a percentage of sequenceidentity with the sequence complementary of said target sequence of atleast about 70%, preferably at least about 80%, more preferably at leastabout 90%, most preferably at least about 95%.

Said nucleic acid according to the invention may be an oligonucleotide.

Preferably, said nucleic acid or oligonucleotide is complementary to aregion of the KIAA1840 gene that contains at least one of the identifiedmutations.

In one embodiment of this aspect of the invention, the isolated nucleicacid according to the invention consists of at least 10 nucleotides,preferably 20 nucleotides, more preferably 40 nucleotides.

Such a nucleic acid according to the invention may advantageously beused as a primer or probe.

A further object of the present invention relates to an isolated nucleicacid, which comprises or consists in a KIAA1840 gene coding sequence(SEQ ID NO:1) that contains one or several mutation(s) selected from thegroup consisting of

-   -   the substitutions: c.6100C>T, c.2198T>G, c.118C>T, c.1235C>G,        c.2833A>G, c.1951C>T, c.869+1G>A, c. 1679 C>G, c.2316+1G>A,        c.2444G>T, c.2444+1G>C, c.2697G>A, c.5470C>T, c.5870C>G,        c.6091C>T, c.6477+4 A>G, c.6856C>T, c.1282A>T c.5974C>T,    -   the deletions: c.529-533delATATT, c.6451delG, c.6832_(—)6833        delAG, c.1203delA, c.1549_(—)1550 delCT, c.6737_(—)6740 delTTGA,        c.1471_(—)1472 delCT, c.1692delA, c.2716delC, c.1668delT,        c.704_(—)705delAT, c. 5989_(—)5992delCTGT, c. 5532_(—)5533delCA,        c.5769delT, c.6739_(—)6742delGAGT, c.4307_(—)4308 delAA c.        733_(—)734delAT, and    -   the insertions: c.7029_(—)7030insT, c.2850_(—)2851insT,        c.3741_(—)3742insA, c. 5982_(—)5983insCTCT, c.        5986_(—)5987insT, c. 3075_(—)3076insA c.2842_(—)2843insG        or a sequence complementary thereto.

In one embodiment of this aspect of the invention, the isolated nucleicacid according to the invention consists of at least 10 nucleotides,preferably 20 nucleotides, more preferably 40 nucleotides.

In another embodiment, the invention relates to an isolated polypeptidewhich comprises the polypeptide sequence of KIAA1840 containing one orseveral mutation(s) selected from the group consisting of p.Q40X,p.I177_F178delfsX178, p.H235RfsX246, p.M245VfsX246, p.K401KfsX415,p.S412X, p.K428X, p.L491DfsX556, p.L517LfsX556, p.F556LfsX577, p.S560X,p.V564VfsX577, p.R651X, p.L733X, p.R815M, p.W899X, p.Q906SfsX920,p.R945G, p.R945GfsX950, p.L950FfsX953, p.V948GfsX953, p.E1026RfsX1029,p.P1248TfsX1264, p.Q1436RfsX1442, p.R1824X, p.S1844SfsX1857,p.S1923RfsX1950, p.S1957X, p.R1992X, p.L1995LfsX2000, p.C1996LfsX1999,p.L1997_(—)1998 delfsX2056, p.R2031X, p.R2034X, p.A2151 PfsX2172,p.I2246_E2247delfsX2260, p. E2247_S2248delfsX2260, p.S2278LfsX2338,p.R2286X and p.V2344CfsX2349.

Diagnostic Method

The inventors have further shown that KIAA1840mutants are associatedwith a hereditary spastic paraplegias (HSP) which is characterized byweakness, spasticity and often loss of vibration sense in the lowerlimbs. More particular, the inventors have shown that KIAA1840 mutationsas above described correlate in all patients with mild mentalimpairment, a thin corpus callosum (TCC) (AR-HSP-TCC) and frequentpolyneuropathy (72% of the patients) in a series of 45 families with thefull clinical criteria of SPG11. In the 2 other kindreds, cerebralimaging was not available to verify the presence of a thin corpuscallosum (TUN2 and TUN14).

Therefore the invention provides an ex vivo method of diagnosing orpredicting a hereditary spastic paraplegia (HSP) in a subject, whichmethod comprises detecting a mutation in the KIAA1840 gene or protein(spatacsin), as compared to a control population, wherein the presenceof a mutation is indicative of an hereditary spastic paraplegia (HSP).

Nucleic Acids Assays:

According to a first embodiment the mutations may be detected byanalysing a KIAA1840 nucleic acid molecule. In the context of theinvention, KIAA1840 nucleic acid molecules include mRNA, genomic DNA andcDNA derived from mRNA. DNA or RNA can be single stranded or doublestranded. These may be utilized for detection by amplification and/orhybridization with a probe, for instance.

Thus the invention provides an ex vivo method of diagnosing orpredicting a hereditary spastic paraplegia (HSP), in a subject, whichmethod may comprise the step consisting of detecting a KIAA1840 mutationin a nucleic acid sample obtained from the subject, wherein the presenceof a mutation is indicative of a hereditary spastic paraplegia (HSP).

The nucleic acid sample may be obtained from any cell source or tissuebiopsy. Non-limiting examples of cell sources available include withoutlimitation blood cells, buccal cells, epithelial cells, fibroblasts, orany cells present in a tissue obtained by biopsy or post-mortem. Cellsmay also be obtained from body fluids, such as blood, plasma, serum,lymph, etc. DNA may be extracted using any methods known in the art,such as described in Sambrook et al., 1989. RNA may also be isolated,for instance from tissue biopsy, using standard methods well known tothe one skilled in the art such as guanidiumthiocyanate-phenol-chloroform extraction (Chomocyznski et al., 1987).

A KIAA1840 mutation according to the invention may be found and locatedin many exons, including exon 1 and exon 39 (Table 2).

KIAA1840 mutations may be detected in a RNA or DNA sample, preferablyafter amplification. For instance, the isolated RNA may be subjected tocoupled reverse transcription and amplification, such as reversetranscription and amplification by polymerase chain reaction (RT-PCR),using specific oligonucleotide primers that are specific for a mutatedsite or that enable amplification of a region containing the mutatedsite. According to a first alternative, conditions for primer annealingmay be chosen to ensure specific reverse transcription (whereappropriate) and amplification; so that the appearance of anamplification product be a diagnostic of the presence of a particularKIAA1840 mutation. Otherwise, RNA may be reverse-transcribed andamplified, or DNA may be amplified, after which a mutated site may bedetected in the amplified sequence by hybridization with a suitableprobe or by direct sequencing, or any other appropriate method known inthe art. For instance, a cDNA obtained from RNA may be cloned andsequenced to identify a mutation in KIAA1840 sequence.

Actually numerous strategies for genotype analysis are available(Antonarakis et al., 1989 Cooper et al., 1991 Grompe, 1993). Briefly,the nucleic acid molecule may be tested for the presence or absence of arestriction site. When a base substitution mutation creates or abolishesthe recognition site of a restriction enzyme, this allows a simpledirect enzymatic test for the mutation. Further strategies include, butare not limited to, direct sequencing, restriction fragment lengthpolymorphism (RFLP) analysis; hybridization with allele-specificoligonucleotides (ASO) that are short synthetic probes which hybridizeonly to a perfectly matched sequence under suitably stringenthybridization conditions; allele-specific PCR; PCR using mutagenicprimers; ligase-PCR, HOT cleavage; denaturing gradient gelelectrophoresis (DGGE), temperature denaturing gradient gelelectrophoresis (TGGE), single-stranded conformational polymorphism(SSCP) and denaturing high performance liquid chromatography (DHPLC)(Kuklin et al., 1997). Direct sequencing may be accomplished by anymethod, including without limitation chemical sequencing, using theMaxam-Gilbert method by enzymatic sequencing, using the Sanger methodmass spectrometry sequencing sequencing using a chip-based technology(see e.g. Little et al., 1996); and real-time quantitative PCR.Preferably, DNA from a subject is first subjected to amplification bypolymerase chain reaction (PCR) using specific amplification primers.However several other methods are available, allowing DNA to be studiedindependently of PCR, such as the rolling circle amplification (RCA),the InvaderTMassay, or oligonucleotide ligation assay (OLA). OLA may beused for revealing base substitution mutations. According to thismethod, two oligonucleotides are constructed that hybridize to adjacentsequences in the target nucleic acid, with the join sited at theposition of the mutation. DNA ligase will covalently join the twooligonucleotides only if they are perfectly hybridized (Nickerson etal., 1990).

The inventors designed a series of primers, manually or using Oligo6(MBI, Cascade, Colo.), in order to amplify all coding exons of 18 genesfrom the candidate interval (primers and conditions available onrequest), including the mutated KIAA1840 gene (see Table 4).PCR-amplified fragments of genomic DNA were then purified usingexonuclease 1 (New England Biolabs, 2 U/5 μl PCR product) and shrimpalkaline phosphatase (Roche, 1 U/5 μl of PCR product) and sequencedusing the fluorescent dideoxy-terminator method (BigDye v3, AppliedBiosystem) on an automated ABI-3730 sequencer according to themanufacturer's recommendations. With the use of the software packageSeqScape (Applied Biosystems), sequences were aligned and compared toconsensus sequences.

Protein Assays

According to a second embodiment said mutation may be detected inKIAA1840 protein or a truncated form of the KIAA1840 protein may bedetected, as compared to a control population.

All of the identified mutations of the KIAA840 gene create somedeletions of the C-terminal part of the spatacsin protein, in some casesbecause of aberrant splicing (FIG. 5). These deletions result intruncated proteins of sequences SEQ ID NO: 149 to SEQ ID NO:164 and SEQID NO:166 to SEQ ID NO:188, respectively. Those due to aberrantsplicing, either very likely, could not be precised because themodification of the splicing could not be evidenced in mRNA directly,except in family FSP670 (r.2834+1_(—)2834+65 ins, p.R945GfsX950). It cannot be excluded, however, that a shorten protein fragment may besynthesized due to the activation of new ATGs after the stop codon.

Said mutation may be detected according to any appropriate method knownin the art. In particular a sample, such as a tissue biopsy, obtainedfrom a subject may be contacted with antibodies specific of the mutatedform of KIAA1840 protein, i.e. antibodies that are capable ofdistinguishing between a mutated form of KIAA1840 and the wild-typeprotein (or any other protein), to determine the presence or absence ofa KIAA1840 specified by the antibody. An antibody recognizing the wildtype protein could also be used to check the presence of the protein orits abnormal location or size and could then be used as a diagnostictool as well.

Antibodies that specifically recognize a mutated KIAA1840 protein alsomake part of the invention. The antibodies are specific of mutatedKIAA1840 protein, that is to say they do not cross-react with thewild-type KIAA1840 protein.

A monoclonal or polyclonal antibody recognizing the wild-type KIAA1840protein may be used to detect the presence of the wild-type protein orone of its truncated forms. For instance, an antibody recognizing theN-terminal part of the wild-type KIAA1840 protein may also recognize oneor several truncated forms and can be used to reveal by immunoblotting,the different forms, wild-type and truncated, according to theirmolecular weights. An antibody recognizing the wild-type KIAA1840protein, but not recognizing the truncated forms, can be used forimmunoblotting or in immunoassay as ELISA; in that case, an absence ofsignal reveals the presence of a truncated form in the sample or theabsence of synthesis of a stable protein as compared with a positivecontrol comprising the wild-type KIAA1840 protein.

The antibodies of the present invention may be monoclonal or polyclonalantibodies, single chain or double chain, chimeric antibodies, humanizedantibodies, or portions of an immunoglobulin molecule, including thoseportions known in the art as antigen binding fragments Fab, Fab′,F(ab′)₂ and F(v). They can also be immunoconjugated, e.g. with a toxin,or labelled antibodies.

Whereas polyclonal antibodies may be used, monoclonal antibodies arepreferred for since they are more reproducible in the long run.

Procedures for raising “polyclonal antibodies” are also well known.Polyclonal antibodies can be obtained from serum of an animal immunizedagainst the spatacsin complex, which may be produced by geneticengineering for example according to standard methods well-known by oneskilled in the art. Typically, such antibodies can be raised byadministering mutated KIAA1840 protein or peptides of this proteinsubcutaneously to New Zealand white rabbits which have first been bledto obtain pre-immune serum. The antigens can be injected at a totalvolume of 100 μl per site at six different sites. Each injected materialmay contain adjuvants with or without pulverized acrylamide gelcontaining the protein or polypeptide after SDS-polyacrylamide gelelectrophoresis. The rabbits are then bled two weeks after the firstinjection and periodically boosted with the same antigen three timesevery six weeks. A sample of serum is then collected 10 days after eachboost. Polyclonal antibodies are then recovered from the serum byaffinity chromatography using the corresponding antigen to capture theantibody. This and other procedures for raising polyclonal antibodiesare disclosed by Harlow et al. (1988) which is hereby incorporated inthe references.

A “monoclonal antibody” in its various grammatical forms refers to apopulation of antibody molecules that contains only one species ofantibody combining site capable of immunoreacting with a particularepitope. A monoclonal antibody thus typically displays a single bindingaffinity for any epitope with which it immunoreacts. A monoclonalantibody may therefore contain an antibody molecule having a pluralityof antibody combining sites, each immunospecific for a differentepitope, e.g. a bispecific monoclonal antibody. Although historically amonoclonal antibody was produced by immortalization of a clonally pureimmunoglobulin secreting cell line, a monoclonally pure population ofantibody molecules can also be prepared by the methods of the presentinvention.

Laboratory methods for preparing monoclonal antibodies are well known inthe art (see, for example, Harlow et al., 1988). Monoclonal antibodies(mAbs) may be prepared by immunizing purified mutated KIAA1840 proteininto a mammal, e.g. a mouse, rat, human and the like mammals. Theantibody-producing cells in the immunized mammal are isolated and fusedwith myeloma or heteromyeloma cells to produce hybrid cells (hybridoma).The hybridoma cells producing the monoclonal antibodies are utilized asa source of the desired monoclonal antibody. This standard method ofhybridoma culture is described in Kohler and Milstein (1975).

While mAbs can be produced by hybridoma culture the invention is not tobe so limited. Also contemplated is the use of mAbs produced by anexpressing nucleic acid cloned from a hybridoma of this invention. Thatis, the nucleic acid expressing the molecules secreted by a hybridoma ofthis invention can be transferred into another cell line to produce atransformant. The transformant is genotypically distinct from theoriginal hybridoma but is also capable of producing antibody moleculesof this invention, including immunologically active fragments of wholeantibody molecules, corresponding to those secreted by the hybridoma.See, for example, U.S. Pat. No. 4,642,334 to Reading; PCT PublicationNo.; European Patent Publications No. 0239400 to Winter et al. and No.0125023 to Cabilly et al.

Antibody generation techniques not involving immunisation are alsocontemplated such as for example using phage display technology toexamine naive libraries (from non-immunised animals); see Barbas et al.(1992), and Waterhouse et al. (1993).

Antibodies raised against mutated KIAA1840 protein may be cross reactivewith wild-type KIAA1840 protein. Accordingly a selection of antibodiesspecific for mutated KIAA1840 protein is required. This may be achievedby depleting the pool of antibodies from those that are reactive withthe wild-type KIAA1840 protein, for instance by submitting the raisedantibodies to an affinity chromatography against wild-type KIAA1840protein.

Alternatively, binding agents other than antibodies may be used for thepurpose of the invention. These may be for instance aptamers, which area class of molecule that represents an alternative to antibodies in termof molecular recognition. Aptamers are oligonucleotide or oligopeptidesequences with the capacity to recognize virtually any class of targetmolecules with high affinity and specificity. Such ligands may beisolated through Systematic Evolution of Ligands by EXponentialenrichment (SELEX) of a random sequence library, as described in TuerkC. and Gold L., 1990. The random sequence library is obtainable bycombinatorial chemical synthesis of DNA. In this library, each member isa linear oligomer, eventually chemically modified, of a unique sequence.Possible modifications, uses and advantages of this class of moleculeshave been reviewed in Jayasena S. D., 1999. Peptide aptamers consists ofa conformationally constrained antibody variable region displayed by aplatform protein, such as E. coli Thioredoxin A that are selected fromcombinatorial libraries by two hybrid methods (Colas et al., 1996).

Kits

According to another aspect of the invention, the KIAA1840 mutation isdetected by contacting the DNA of the subject with a nucleic acid probe,which is optionally labeled.

Primers may also be useful to amplify, analyse (dHPLC, Southern . . . )or sequence the portion of the KIAA1840 gene containing the mutatedpositions of interest.

Such probes or primers are nucleic acids that are capable ofspecifically hybridizing with a portion of the KIAA1840 gene sequencecontaining the mutated positions of interest. That means that they aresequences that hybridize with the portion mutated KIAA1840 nucleic acidsequence to which they refer under conditions of high stringency.

The present invention further provides kits suitable for determining atleast one of the mutations of the KIAA1840 gene.

The kits may include the following components:

(i) a probe, usually made of DNA, and that may be pre-labelled.Alternatively, the probe may be unlabelled and the ingredients forlabelling may be included in the kit in separate containers; and

(ii) hybridization reagents: the kit may also contain other suitablypackaged reagents and materials needed for the particular hybridizationprotocol, including solid-phase matrices, if applicable, and standards.

In another embodiment, the kits may include:

(i) sequence determination or amplification primers: sequencing primersmay be pre-labelled or may contain an affinity purification orattachment moiety and

(2) sequence determination or amplification reagents: the kit may alsocontain other suitably packaged reagents and materials needed for theparticular sequencing amplification protocol. In one preferredembodiment, the kit comprises a panel of sequencing or amplificationprimers, whose sequences correspond to sequences adjacent to at leastone of the polymorphic positions, as well as a means for detecting thepresence of each polymorphic sequence.

In a particular embodiment, it is provided a kit which comprises a pairof oligonucleotide primers specific for amplifying all or part of theKIAA1840 gene comprising at least one of the mutated positions that areidentified above (see Table 2).

More preferably, the kits of the invention comprise a pair of primersselected from the pairs shown in Table 3 either for detection by directsequencing or by screening by dHPLC when they could be set-up (secondset of primer pairs).

TABLE 3  Primers used for PCR or for dHPLC (in parentheses) SEQ ID ExonsMutations For/Rev primers NO: 1 c.118C>T, p.Q40Xccacaggaaacgaatggaat/ggttctgtgaggaaaccacg 3/4 3 c.529_533delATATT,cagggacattgtaggccatc/tcccagctcccaaaactaaa 5/7 p.I177_F178delfsX(ccagttgtaaaattgtgacc)/(tcaatcaacacttctaccac) (6)/(8) 4 c.733_734delAT,caggttctttcttgtggcatca/cgaggatatttttaacctcttatca  9/10 p.M245VfsX(gttaggcatacttacaaaactggc)/(cgaggatatttttaacctcttatca) (11)/(12)c.704_705delAT, p.H235RfsX246 c.869+1G>A, r.? 6 c.1203delA, p.K401KfsX;gaacatctttgccctggttt/caggcactgaggcagaagta 13/15 c.1235C>G, p.S412X(ctgtgacaggtgttaagtta)/(atctaatacaagacagtctc) (16) c.1282A>T, p.K428X 7c.1549_1550delCT, aaaaatcaattcctaaatcataatcc/tcttttaaagccaaaaagggtaaa17/19 p.L517LfsX (tagtactgaagtattgagta)/(ttaagtaatgttcttgggca) (20) 8c.1668delT, cttgccccagattgcataat/tccaaaaagtacgtaaaatccca 57/58p.F556LfsX577 c.1679C>G, p.S560X c.1692delA, p.V564VfsX577 10c.1951C>T, p.R651X cccaggactaatcatgaagga/atccccaaaccgataaaacc 21/22 11c.2198T>G, p.L733X cggtgtgtcttccactagctc/acccagccattctcagtgtt 23/25(gttacataaatgtataatccctg)/(cattttaagactttatggattac) (24)/(26) 12c.2316+1G>A, r.? tttgaaagagcagaaagctatgg/tgaaggggttgtcacactttt 61/62 13c.2444G>T, p.R815M ttgtggcaaaagaaaatttgtg/gagaatgcaggctcagttcc 63/64and/or r.? c.2444+1G>C, r.? 15 c.2833A>G,cacagcgagatcctgtctca/cctcactgtaagatgatgccc 27/28 r.2834+1_2834+65ins,p.R945GfsX950 or p.R945G c.2697G>A, p.W899X c.2716delC, pQ906SfsX920 16c.2842_2843insG, cctttaaatactacagtggtgcaga/ccaactgttgagatggagaaaa 29/31p.V948GfsX; (tgtgggcatgatttggtcta)/(acctgctcaaggacaaatgc) (30)/(32)c.2850_2851insT, p.L950FfsX 17 c.3075_3076insA,ttgtttccagatcatgaagaatatg/tcagatagctgaccacagcc 67/68 p.E1026RfsX1029 22c.3741_3742insA, agtcagcttaagggaagcgg/gaagataaccattttctcccca 77/78p.P1248TfsX1264 25 c.4307_4308delAA,aaaaggcaccatacagctttg/ggaaacacatgctggaacct 83/84 p.Q1436RfsX1442 30c.5470C>T, p.R1824X tgaggtgggaggatctcttg/gatgtgttcagagcagccaa 93/94c.5532_5533delCA, and p.S1844SfsX1857taagctggaggagctggaga/ttgttgtccccttaacttgg 95/96 c.5769delT,p.S1923RfsX1950 31 c.5974C>T, p.R1992Xtttgaagtatcccagggtgg/ccaccattccccaaagataa 33/34 c.5870C>G, p.S1957Xc.5982_5983insCTCT, p.L1995LfsX2000 c.5986_5987insT, p.C1996LfsX1999c.5989_5992delCTGT, p.L1997_Y1998>M1997fs X2056 32 c.6100C>T, R2034Xttacctggatttggctttgg/tgcaatccagaaacttgagaga 35/37 c.6091C>T, p.R2031X(cctggcttctaaaagtggcc)/(aagcacaacatccaaatcctt) (36)(38) 34c.6451delG, p.A2151PfsX atgttggcaggaactccatc/ctcctttggagcaacctctg 39/40c.6477+4 A>G, r.? 36 c.6737_6740delTTGA,ttccaacaggaaagcacaca/cagctacttgggaggctgag 41/43 p.I2246_E2247delfsX(caacaggaaagcacacatgc)/(gtgtggctgtgacctcactc) (42)/(44)c.6739_6742delGAGT, p.E2247_S2248>L2247fs X2260 37 c.6832_6833delAG,gcattagaaggggcactgaa/ctcacaacggtattcacccc 45/47 p.S2278LfxX(aacatggctgggatgtttct)/(ttcctggttggcctatgatg) (46)/(48) 38c.6856C>T, p.R2286X ttttgtccttgggctctttc/cctggttctgtcactagccc 101/102 39c.7029_7030insT, aagggtttaagataatttgggga/ggattcttgatactgctttgcc 49/51p.V2344CfsX (aatgccaaacacacacctga)/(ctcaaagcagaggcaaggag) (50)/(52)

Therapeutic Methods

The inventors have demonstrated that the all, except one, mutationsidentified in the KIAA1840 gene cause truncation of the protein,suggesting that pathogenicity results from loss of function.

These results identify mutated KIAA1840 gene as target for thepreventive or curative treatment of a hereditary spastic paraplegia.

Thus the invention further relates to a method of treatment of an HSPwhich comprises the step of administering a subject in need thereof witha KIAA1840 nucleic acid, i.e. a nucleic acid sequence that encodes awild-type KIAA1840 protein, so that spatacsin is expressed in vivo bythe cells of the subject that have been transfected with said nucleicacid. Accordingly, said method leads to an overexpression of wild-typespatacsin which compensates expression of defective mutated KIAA1840protein.

The invention also relates to the use of a KIAA1840 nucleic acid for themanufacture of a medicament intended for the treatment of an HSP.

In the context of the invention, the term “treating” or “treatment”, asused herein, means reversing, alleviating, inhibiting the progress of,or preventing the disorder or condition to which such term applies, orone or more symptoms of such disorder or condition.

Preferably said KIAA1840 nucleic acid is administered in atherapeutically effective amount. A “therapeutically effective amount”is intended for a minimal amount of active agent (e.g., KIAA1840 nucleicacid) which is necessary to impart therapeutic benefit to a subject. Forexample, a “therapeutically effective amount” to a mammal is such anamount which induces, ameliorates or otherwise causes an improvement inthe pathological symptoms, disease progression or physiologicalconditions associated with or resistance to succumbing to a disorder.

The administered polynucleotide comprises the nucleotide sequence SEQ IDNO:1, or any homologous or similar sequence as defined below:

a) a sequence showing at least 70%, preferably at least 75% or 80% or85% or 90% or 95% or 99%, sequence similarity with SEQ ID NO:1;

b) a sequence hybridizing with SEQ ID NO:1, or its complementarysequence, under stringent conditions;

c) a sequence encoding a protein of sequence SEQ ID NO:2, or anysequence substantially similar with SEQ ID NO:2.

The term “sequence similarity” in all its grammatical forms refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences of proteins that may or may not share a commonevolutionary origin. Preferably the degree of sequence identity iscalculated compared with the totality of a reference sequence.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least 70%, preferably atleast 75% or 80% or 85% or 90% or 95% or 99%, of the nucleotides matchover the defined length of the DNA sequences, as determined by sequencecomparison algorithms, such as BLAST, FASTA, DNA Strider, etc. Sequencesthat are substantially homologous can be identified by comparing thesequences using standard software available in sequence data banks, orin a Southern hybridization experiment under, for example, stringentconditions as defined for that particular system.

Similarly, in a particular embodiment, two amino acid sequences are“substantially similar” when greater than 80%, preferably than 85% or90% or 95% or 99%, of the amino acids are similar (functionallyidentical). “Functionally identical” polypeptides are those in which agiven amino acid residue has been changed without altering the overallconformation and function of the polypeptide, including, but not limitedto, replacement of an amino acid with one having similar properties(such as, for example, polarity, hydrogen bonding potential, acidic,basic, hydrophobic, aromatic, and the like). Amino acids with similarproperties are well known in the art. For example, arginine, histidineand lysine are hydrophilic-basic amino acids and may be interchangeable.Similarly, isoleucine, a hydrophobic amino acid, may be replaced withleucine, methionine or valine. Such changes are expected to have littleor no effect on the apparent molecular weight or isoelectric point ofthe protein or polypeptide. Preferably, the similar sequences areidentified by alignment using, for example, the GCG (Genetics ComputerGroup, Program Manual for the GCG Package, Version 7, Madison, Wis.)pileup program, or any of the programs described above (BLAST, FASTA,etc.).

Preferably the KIAA1840 nucleic acid sequence according to the inventionis associated with elements that enable for regulation of itsexpression, such as a promoter sequence.

Such a nucleic acid may be in the form of a DNA vector. The terms“vector” means the vehicle by which a DNA or RNA sequence (e.g. aforeign gene) can be introduced into a host cell, so as to transform thehost and promote expression (e.g. transcription and translation) of theintroduced sequence. A common type of vector is a “plasmid”, whichgenerally is a self-contained molecule of double-stranded DNA, usuallyof bacterial origin, that can readily accept additional (foreign) DNAand which can readily introduced into a suitable host cell. A plasmidvector often contains coding DNA and promoter DNA and has one or morerestriction sites suitable for inserting foreign DNA.

The KIAA1840 nucleic acid may be introduced into a target cell by meansof any procedure known for the delivery of nucleic acids to the nucleusof cells, ex vivo, on cells in culture or removed from an animal or apatient, or in vivo.

Ex vivo introduction may be performed by any standard method well knownby one skilled in the art, e.g. transfection, electroporation,lipofection, microinjection, transduction, cell fusion, DEAE dextran,calcium phosphate precipitation, or use of a gene gun.

The above methods do not limit the scope of the invention and it is tobe understood that the one skilled in the art may readily make use ofany other known appropriate methods for delivering a nucleic acid to acell in vivo or in vitro.

The invention also relates to the use of wild-type KIAA1840 protein(spatacsin) for the manufacture of a medicament intended for thetreatment of an HSP.

Thus the invention further relates to a method of treatment of an HSPwhich comprises the step of administering a subject in need thereof witha therapeutically effective amount of wild-type KIAA1840 protein.

The KIAA1840 protein may be introduced to a target cell by means of anyprocedure known for the delivery of proteins to cells, ex vivo, on cellsin culture or removed from an animal or a patient, or in vivo.

Protein delivery is the process by which a protein crosses the cellplasma membrane. Traditionally, methods to introduce antibodies,peptides or other membrane-impermeable molecules into cells includemicro-injection and electroporation.

A number of protein-transduction domains (PTDs) have also been developedthat mediate protein delivery into cells. These PTDs or signal peptidesequences are naturally occurring polypeptides of 15 to 30 amino acids,which normally mediate protein secretion in the cells. They are composedof a positively charged amino terminus, a central hydrophobic core and acarboxyl-terminal cleavage site recognized by a signal peptidase.Examples of such membrane-transducing peptides include Trojan peptides,human immunodeficiency virus (HIV)-1 transcriptional activator (TAT)protein or its functional domain peptides, and other peptides containingprotein-transduction domains (PTDs) derived from translocation proteinssuch as Drosophilia homeotic transcription factor Antennapedia (Antp)and herpes simplex virus DNA-binding protein, VP22, and the like. Somecommercially available peptides, for example, penetratin 1, Pep-1(Chariot reagent, Active Motif Inc., CA) and HIV GP41 fragment(519-541), can be used for protein delivery.

Recently, the use of lipid liposomes or the like that can complex with aprotein of interest and promote the delivery of the protein into thecell has also been demonstrated. Products available commercially can beused, such as BioPORTER (Gene Therapy Systems), or ProVectin (Imgenex,San Diego, Calif.).

The above methods do not limit the scope of the invention and it is tobe understood that the one skilled in the art may readily make use ofany other known appropriate methods for delivering a protein to a cellin vivo or in vitro.

The invention will be further illustrated by the following figures andexamples.

FIGURES

FIGS. 1 and 2: Multipoint linkage analysis performed in 16 families for34 microsatellite markers from chromosome 15q. cM=centimorgan.

(FIG. 1) Multipoint LOD score values for each family. *Relative positionon the genetic map of chromosome 15 (according tohttp://research.marshfieldclinic.org/genetics).

(FIG. 2) Cumulative multipoint LOD scores in the 16 linked-familiesplotted according to the genetic map of chromosome 15.

FIGS. 3 and 4: Pedigrees of 2 SPG11 families that reduced the candidateinterval. Black circles (women) and squares (men) indicate affectedmembers compared to previous publications. The numbers are an internalreference for each sampled individual. Asterisks indicate sampledsubjects. Haplotype reconstruction for selected microsatellite markerspositioned according to the human genome draft sequence(www.ncbi.nlm.nih.gov, www.ensembl.org) is shown. The homozygoushaplotype, in which the mutated gene has been located, is flanked byblack boxes. Arrowheads indicate the position of probable recombinationevents. cM=centimorgan (according tohttp://research.marshfieldclinic.org/genetics).

FIG. 5: Critical region of SPG11. (a) Physical map of human chromosome15q15-21 with selected genetic markers and candidate genes that weresequenced. Distances in megabases are indicated relative to chromosome15 according to the Ensembl database. Markers defining the reducedcandidate interval are in bold. # indicates that these genes (SEMA6D andMAP1A) were analyzed in a previous study (Stevanin et al, 2006). > and <indicate the orientation of the open reading frame (ORF) of each gene.(b) Exon-intron structure of the 101 Kb of the KIAA1840 gene, also knownas FLJ21439, with positions of mutations identified in 17 SPG11families. (c) Putative functional domains (boxes) and their positions onthe protein sequence. TM=transmembrane domains. Regions I and IIcorrespond to structurally similar domains based on their hydrophobicitystatus analysed with DomHCA software.

FIGS. 6 to 17: Pedigrees and segregation of the 17 mutations detected inKIAA1840. Square symbols are men, the circles are women. The filledsymbols are affected individuals, grey or ? symbols indicate patientswith an unknown status. The numbers are an internal reference for eachsampled individual. Stars indicate sampled subjects. M or m=mutation;+=wild type. Electrophoregrams are shown for the homozygous mutationsonly. (6,7) Families with common origins sharing the same mutations.Haplotypes for three close microsatellites segregating with themutations are highlighted. The correspondence between the numbering ofalleles and their size in base pairs is indicated. (8, 10 to 14) Otherhomozygous mutations. (15) New homozygous mutations. (9, 16, 17)Compound heterozygous mutations.

FIG. 18: Internal structural duplication in spatacsin

(a) Hydrophobic cluster analysis (HCA) plots of the internal duplicationof two regions in the human sequence. The HCA method is based on the useof a bidimensional plot (HCA plot) from the drawing of the 1D sequenceon an alpha helix (3.6 residue/turn, connectivity distance of 4 residuesseparating two different clusters) which has been shown to offer thebest correspondence between clusters and regular secondary structures.Examination of the HCA plot of a protein sequence enables globularregions to be easily distinguished from non globular ones and, inglobular regions, secondary structures to be identified. This 2Dsignature, which is much more highly conserved than the 1D sequence andcan be enriched from the comparison of families of highly divergentsequences, enables relevant similarities to be successfully detected atlow levels of sequence identity. The form of the clusters is generallyindicative of the type of secondary structures (vertical clusters areoften associated with beta strands whereas horizontal ones oftencorrespond to alpha helices). DomHCA software:http://www.lmcp.jussieu.fr/%7Emornon/hca.html. Special symbols are usedfor some amino acids: star for proline, square and dotted square forthreonine and serine, diamond for glycine.

(b) Multiple alignment of the structural repeat domains (I and II, FIG.3) corresponding to the HCA plots (DomHCA software). Under the multiplealignment, highly conserved residues are indicated by a capital letterwhen strictly conserved or in lower case if there is some homology. Thecharacter “&” means that this position is always occupied by ahydrophobic residue (amino acids FILMVW and Y).

FIG. 19: Expression profile of KIAA1840 examined by northern blot inhuman adult brain. The transcripts were present in all brain tissues.Note the 8-Kb transcript more intensely expressed in the cerebellumwhile the 5.5-Kb transcript is mainly found in the cerebral cortex.

Table 1: Exon-intron boundaries in the KIAA1840 gene

Table 2: Mutations found in the KIAA1840 gene in families withAR-HSP-TCC.

Table 3:Primers used for detecting the mutations either by directsequencing or by dHPLC

Table 4: Primers used for the amplification of all exons of the KIAA1840gene and PCR amplification conditions

Table 5: PCR conditions and dHPLC conditions to analyze exons ofKIAA1840.

Table 6: dHPLC primers to analyze exons of KIAA1840

EXAMPLE Method

Subjects: 211 individuals, including 83 affected members and 44 nonmutated members, from 91 families.

All patients were examined by a neurologist. They were selected among216 families with hereditary spastic paraparesis compatible withrecessive transmission collected in our neurogenetic reference center incollaboration with the SPATAX network. They presented a typical “SPG11”phenotype defined as the presence of progressive spastic paraparesisassociated with thin corpus callosum on cerebral MRI and mentalretardation and neuropathy.

Blood samples was obtained after written consent from all affectedpatients and their relatives with approval of the local Ethic committeeof Paris-Necker (approval n^(o) 03.12.07 of the comité Consultatif pourla Protection des Personnes et la Recherche Biomédicale, to A.D).Genomic DNA was extracted from leukocytes using standard procedures.

Linkage analysis: The genome scan in family FSP221 was performed using400 microsatellites, regularly spaced on all chromosomes (ABI-Prismmapping set v2, Applied Biosystems, Foster City, Calif.) and 50additional polymorphic markers were used to analyze the results.Genotypes were determined by PCR with a fluorescently-labeled primer,electrophoretic migration in an ABI-3730 sequencer (Applied Biosystems)and analysis with Genescan 3.5 (Applied Biosystems). Allegro 1.2c wasused to calculate two-point and multipoint lod scores between thedisease phenotype and each of the markers or the map of the markersassuming a complete penetrance, equal allele frequencies for the markersand a mutated allele frequency of 0.0005 (Gudbjartsson et al. 2000).Marker order and genetic distances were obtained from the Ensembl(http://www.ensembl.org) and Marshfield databases(http://research.marshfieldclinic.org/genetics), respectively.

Mutation Detection:

A series of primers was designed manually or using Oligo6 (MBI, Cascade,Colo.) in order to amplify all coding exons of 18 genes from thecandidate interval (primers and conditions available on request).PCR-amplified fragments of genomic DNA were then purified usingexonuclease 1 (New England Biolabs, 2 U/5 μl PCR product) and shrimpalkaline phosphatase (Roche, 1 U/5 μl of PCR product) and sequencedusing the fluorescent dideoxy-terminator method (BigDye v3, AppliedBiosystem) on an automated ABI-3730 sequencer according to themanufacturer's recommendations. With the use of the software packageSeqScape (Applied Biosystems), sequences were aligned and compared toconsensus sequences.

Primers used for the amplification of the KIAA1840 gene are listed inthe following Table 4.

The conditions of the PCR programme are as follows:

95° C., 12 min

then 40 cycles of:

-   -   95° C., 30 s    -   Annealing Temperature (see Table 4), 30 s,    -   72° C., 30 s

then

72° C., 10 min, and

15° C., 15 min.

PCR were performed in 25 μl final volume using 10 pmol of each primer,at final concerntrations of 1.5 mM MgCl2 and 0.24 mM dNTP.

Taq pol, which is commercially available from Quiagen was used exceptfor exons 6, 12 and 40B where Taq GOLD (Applied Biosystems) was used.

TABLE 4  Annealing SEQ ID Exon temperature NO: Forward primerReverse primer ex1 60° C. 3/4 ccacaggaaacgaatggaat ggttctgtgaggaaaccacgex2 60° C. 53/54 ctgagccccacatttttgtt caagtgctcaatagccccat ex3 60° C.5/7 cagggacattgtaggccatc tcccagctcccaaaactaaa ex4 60° C.  9/10caggttctttattgtggcatca cgaggatatttttaacctcttatca ex5 62° C. 55/56gctaactgcccttaatagagtaaaa aaagggtacagcgtcagcat ex6 TD62-58 13/15gaacatctttgccctggttt caggcactgaggcagaagta ex7 60° C. 17/19aaaaatcaattcctaaatcataatcc tcttttaaagccaaaaagggtaaa ex8 60° C. 57/58cttgccccagattgcataat tccaaaaagtacgtaaaatccca ex9 60° C. 59/60cagcaaaagggtaatagcagtg cccaaatgtagtaaatggcg ex10 60° C. 21/22cccaggactaatcatgaagga atccccaaaccgataaaacc ex11 60° C. 23/25cggtgtgtcttccactagctc acccagccattctcagtgtt ex12 TD62-58 61/62tttgaaagagcagaaagctatgg tgaaggggttgtcacactttt ex13 60° C. 63/64ttgtggcaaaagaaaatttgtg gagaatgcaggctcagttcc ex14 60° C. 65/66atgtggaactgagcctgcat cgacttgcattttaaagaacctg ex15 60° C. 27/28cacagcgagatcctgtctca cctcactgtaagatgatgccc ex16 62° C. 29/31cctttaaatactacagtggtgcaga ccaactgttgagatggagaaaa ex17 56° C. 67/68ttgtttccagatcatgaagaatatg tcagatagctgaccacagcc ex18 60° C. 69/70tccctcttaaggagaaaaacactg accgggccgagatataaaat ex19 60° C. 71/72gctagtttgtcttagaaccagaaca ttttgggttgtctcactatcaca ex20 60° C. 73/74aaggaacatagccagttctgtttt tgcgaactatttttcctttgg ex21 60° C. 75/76tgcaacttctcaggtacacatct aggctagagtgcagtggcat ex22 60° C. 77/78agtcagcttaagggaagcgg gaagataaccattttctcccca ex23 60° C. 79/80ttgtgagtgtttggggagaa ggggatttagtgaaaacacca ex24 64° C. 81/82tttgttggagaatacactgtgctt catgtctacacaacagaaagaatgc ex25 60° C. 83/84aaaaggcaccatacagctttg ggaaacacatgctggaacct ex26C 55° C. 85/86cttctgtctgcttcttggtctt tatcatcattatctgttgttgg ex27 60° C. 87/88ttaggtgatcccactggctc cccaggagttcaaggctgta ex28 60° C. 89/90ctgaggagggcttgtttttg tctgtaacttgtttactcccagttg ex29 60° C. 91/92gatcacaccactgcattcca ggcacctgtagtcccagcta ex30A 60° C. 93/94tgaggtgggaggatctcttg gatgtgttcagagcagccaa ex30B 60° C. 95/96taagctggaggagctggaga ttgttgtccccttaacttgg ex31 60° C. 33/34tttgaagtatcccagggtgg ccaccattccccaaagataa ex32 60° C. 35/37ttacctggatttggctttgg tgcaatccagaaacttgagaga ex33 60° C. 97/98caataggccaagggtttcaa tataactcctgctggagggc ex34 62° C. 39/40atgttggcaggaactccatc ctcctttggagcaacctctg ex35 60° C.  99/100ggtagcctggaaattagccc tgaaccagaatctgaagcca ex36 62° C. 41/43ttccaacaggaaagcacaca cagctacttgggaggctgag ex37 60° C. 45/47gcattagaaggggcactgaa ctcacaacggtattcacccc ex38 60° C. 101/102ttttgtccttgggctctttc cctggttctgtcactagccc ex39 60° C. 49/51aagggtttaagataatttgggga ggattcttgatactgctttgcc ex40A 60° C. 103/104aattagccagggtggtgaca cccacaaaggactgatatgg ex40B TD62-58 105/106aaggaccctcagacaggttg tcctttaaggcagacaaggg TD = TouchDown 10 cyclesdecrease of annealing temperature, then 25 stable cycles. Temperaturesin Celsius degrees.

For some exons, it was possible to set up dHPLC conditions to detectvariants. Primers different from those used for direct sequencing werespecifically designed but they can also be used for direct sequencing.The PCR conditions and dHPLC conditions are indicated on table 5.

TABLE 5 dHPLC conditions to analyze exons of KIAA1840. Temperature inCelsius degrees. Exon Size T° PCR T° DHPLC 2 323 62° - 1′ - 35x 55.3° 3305 58° - 1′ - 35x 55.1° 4 320 62° - 1′ - 35x 54.8°-52.8° 5 330 60° -1′ - 35x 54.9° 6 450 58° - 1′ - 35x 54°-53° 7 275 58° - 1′ - 35x50.6°-52.6° 9 342 62° - 1′ - 35x 54.1  11 293 57° - 1′ - 35x 54.6° 12210 62° - 1′ - 35x 52.5° 13 289 62° - 1′ - 35x 51.5° 14 246 62° - 1′ -35x 55.8° 16 309 62° - 1′ - 35x 55.2° 17 239 62° - 1′ - 35x 53.9° 18 32458° - 1′ - 35x 53°-50° 20 311 62° - 1′ - 35x 52.3° 22 383 62° - 1′ - 35x55.8° 23 356 62° - 1′ - 35x 53.1° 24 267 60° - 1′ - 35x 57.1° 25 36160° - 1′ - 35x 56.6° 27 330 62° - 1′ - 35x 53.6° 28 329 62° - 1′ - 35x53.5° 29 330 56° - 1′ - 35x 54.2°-56.2° 32 323 60° - 1′ - 35x 58.8° 33349 62° - 1′ - 35x 57.6° 35 312 62° - 1′ - 35x 54°   36 376 62° - 1′ -35x 52°   37 313 62° - 1′ - 35x 57.6° 38 315 62° - 1′ - 35x 56.9° 39 38062° - 1′ - 35x 53.2° 40 390 62° - 1′ - 35x 54.4° 40 321 58° - 1′ - 35x54.2°

TABLE 6  dHPLC primers to analyze exons of KIAA1840 ExonPRIMERS F (5′-3′)/R (5′-3′) SEQ ID NO: 2accaggtcaactaaactgttctct/tatgctgaaagaccacctgtaga 107/108 3ccagttgtaaaattgtgacc/tcaatcaacacttctaccac 6/8 4gttaggcatacttacaaaactggc/cgaggatatttttaacctcttatca 11/12 5caggagcagtagtaacacaa/aaagggtacagcgtcagcat 109/110 6ctgtgacaggtgttaagtta/atctaatacaagacagtctc 14/16 7tagtactgaagtattgagta/ttaagtaatgttcttgggca 18/20 9gcaggtaataagcctgcagaa/cccccttcctagctgctatt 111/112 11gttacataaatgtataatccctg/cattttaagactttatggattac 24/26 12tgttcaaaatagttccattacaaaa/tttcttccaaggttttcttcca 113/114 13tttgcaaaagtgcttgatttt/tgcaggctcagttccacata 115/116 14ggaatgatgcctttttctcc/tctcacacttgccttctgga 117/118 16tgtgggcatgatttggtcta/acctgctcaaggacaaatgc 30/32 17aatcatcgcctgagcaaaat/ccagtgactgatccaaagca 119/120 18ccctcttaaggagaaaaacac/cagccttatcctctgctctt 121/122 20tggaaaaggggagcagacta/tgcgaactatttttcctttgg 123/124 22gaggaggccacaaatcacat/gccttagacctcgtcacacc 125/126 23tgctcaggttttgactttttctc/tttcactgatggcaagatgc 127/128 24accacccccacctctaattc/ctacacaacagaaagaatgc 129/130 25ccagctgaaactgaaagttgg/ctgggtacttacttcaggct 131/132 27cactgtgccctgccttatta/tgtgcctgagtaaccgagtg 133/134 28tcccagatttggaggttttg/tgcattttaatttcctaactaccc 135/136 29gctgtagtggcattttattg/cctgggtgacagagcaagac 137/138 32cctggcttctaaaagtggcc/aagcacaacatccaaatcctt 36/38 33agctgcagagctccataagc/taggcatccagagcaggaac 139/140 35ggcatctgaaagcaaccact/ccctccattttcccaagagt 141/142 36caacaggaaagcacacatgc/gtgtggctgtgacctcactc 42/44 37aacatggctgggatgtttct/ttcctggttggcctatgatg 46/48 38ggggtgaataccgttgtgag/acctctgggttccatgagtg 143/144 39aatgccaaacacacacctga/ctcaaagcagaggcaaggag 50/52 40agactgctcctctgcactcc/ccgggattgttcaactttagc 145/146 40cagtatcttaacctgtacat/ccgggattgttcaactttagc 147/148

Overexpression Studies:

The KIAA1840 cDNA from clone pf01011 (Kazusa DNA research Institute,Japan) was excised from the pBluescript II SK(+) vector using XhoI/NotIrestriction enzymes and cloned in fusion with EGFP in a SalI/Bsp120Idigested pEGFP-C1 vector (Clontech). The construction was verified bydirect sequencing after ligation, transformation and plasmid extractionusing standard procedures.

COS-7 cells were maintained in DMEM (Invitrogen) supplemented with 10%fetal bovine serum, penicillin (100 UI/ml) and streptomycin (100 μg/ml).Cells were plated 24 h before transfection on cover slips coated withpolyethylenimine and transfected with Lipofectamine-PLUS reagentsaccording to the manufacturer's instructions (Invitrogen). For 6-wellplates, 1-2 μg of plasmid DNA was used per well. Cells were analyzed byimmunofluorescence 120 h post-transfection. The spatacsin-EGFP fusionprotein was observed directly after fixation for 15 min with 4%formaldehyde. Immunocytochemistry was performed, using classicalprocedures with the following antibodies: rabbit anti-Cox2 (1/200, kindgift of A. Lombes, Paris) and rabbit anti-alpha-COP (1/1000^(e);Affinity Bioreagent). Cells were counterstained with DAPI (1 μg/ml,Sigma) and mounted with Fluoromount-G (Southern Biotech). Samples wereobserved with a Leica SP1 confocal microscope. Leica confocal softwarewas used to acquire the images.

Northern-Blot analysis (Human): Total RNA was extracted from the humanpost-mortem brain cortex of an healthy individual (Brain Bank of INSERMU679) using the RNAeasy Mini kit (Qiagen). The corresponding cDNAs weresynthesized using random hexamers in the presence of Thermoscriptreverse transcriptase as recommended by the supplier (Invitrogen). Aseries of 7 probes of 1.2 Kb covering the entire KIAA1840 cDNA wasamplified by PCR at an annealing temperature of 60° C. (primer sequencesavailable on request). Human multiple tissue northern blots (Clontech)were hybridized at 68° C. for 1 hour with a mix of these probesaP³²-labeled by random priming (Prime-it II Random Primer Labeling kit,Stratagene) and purified using ProbeQuant G-50 micro columns (AmershamBiosciences) in accordance to the manufacturer's recommendations toreach a specific activity of at least 1×10⁹ cpm/μg. Membranes were thenwashed as recommended by Clontech then exposed to X-Ray film forautoradiography.

In Situ Hybridization (Rat):

Young (P1, P6, P15 and P21, n=1 each) and adult (P68, 200 g, n=2)Sprague Dawley rats (Charles River) were killed by decapitation andtheir brains were rapidly extracted and frozen in isopentane at −50° C.Sections were prepared with a cryostat at −20° C., from medulla tostriatum (+1.7 from bregma) 600 μm-spaced, thaw-mounted on glass slidesand stored at −80° C. until usage. KIAA1840 mRNA expression was analyzedusing 3 antisens oligonucleotides designed using Helios ETC oligo designsoftware (Helios Biosciences, Paris, France) on the mRNA sequence(XM-242139) of Rattus norvegicus similar to hypothetical proteinFLJ21439 (LOC311372). Each oligonucleotide or a mix of the 3oligonucleotides were used for the hybridization step and gave identicalresults. A mix of three sens oligonucleotides was used as a negativecontrol.

In situ hybridization was performed as described in Moutsimilli et al.(2005) Briefly, oligonucleotides were labeled with [35S]-dATP (AmershamBiosciences) using terminal transferase (Amersham Biosciences) to aspecific activity of 5×10⁸ dpm/μg. The day of the experiment, slideswere fixed in 4% formaldehyde in PBS, washed with PBS, rinsed withwater, dehydrated in 70% ethanol and air-dried. Sections were thencovered with 140 μl of hybridization medium (Helios Biosciences, Paris,France) containing 3-5×10⁵ dpm of the labeled oligonucleotide mix.Slides were incubated overnight at 42° C., washed and exposed to aBAS-SR Fujifilm Imaging Plate for 5-10 days. The plates were scannedwith a Fujifilm Biolmaging Analyzer BAS-5000 and analyzed with MultiGauge Software (Fuji).

For double labeling experiments, brains were processed as for in situhybridization. After the last wash step, sections were fixed in 4%paraformaldehyde in PBS and preincubated in PBS containing 6% goat serumand 0,1% triton. Sections were next incubated with mouse antibodiesdirected against Neu-N(Chemicon International, 1/250), in the samebuffer, processed with biotinylated horse anti-mouse IgG antibodies andABC reagents (Vector Laboratories, Burlingame, Calif.) and submitted toemulsion autoradiography. The labeling with the antisense probe incomparison with the Neu-N neuronal specific counterstaining wasobserved.

Bioinformatics:

Functional domains were searched using bioinformatics tools availableonline at BABEL (http://babel.infobiogen.fr:1984/), Ressource Parisienneen Bioinformatique Structurale (http://bioserv.rpbs.jussieu.fr/RPBS) andPSORT (http://psort.nibb.ac.jp/). Psi-blast (www.ncbi.nlm.nih.gov) wasused to look for homologous proteins or peptides. Alignment ofhomologous proteins was performed using CLUSTALW(http://www.ebi.ac.uk/clustalw/). Alteration of splicing sites wasverified in the Alternative Splicing Database athttp://rulai.cshl.edu/new_alt_exon_db2/HTML/score.html.

HCA is a method that allows to represent a protein sequence on abidimensional scaffold that increases the density of the amino acids,and consequently, evidences local compacity of hydrophobic residues.They form clusters according to a connectivity that is the one of analpha helix. It has been shown that the centers of the clusters and thecenters of the secondary structures statistically match, (Woodcocks etal. 1992) and on the other hand the shape of a cluster is related to thenature of the secondary structure (Callebaut et al. 1997). HCA is a veryefficient tool for recovery of highly divergent internal duplication ofdomains and for the detection of globular domain limits.

Results:

We selected a series of 91 families of European or North-Africanorigins, all without mutations in the SPG7 gene and with a typicalAR-HSP-TCC phenotype. Six of these families were previously reported aslinked to SPG11 using a subset of polymorphic markers from the interval(Casali et al, 2004; Stevanin et al, 2006; Lossos et al, 2006). Theother families were new. All available family members of 16 mostinformative families were genotyped using 34 microsatellite markers forlinkage to three successive loci on chromosome 15 which have beenassociated with thin corpus callosum; SPG11, SPG21 and the locus foragenesis of corpus callosum with polyneuropathy (ACCPN). Positivemultipoint LOD scores ranging from 0.60 to 3.85 and corresponding to themaximal expected values in the pedigrees were obtained in the 16 mostinformative families (FIGS. 1 and 2). Mutations in the ACCPN or SPG21loci were excluded by direct sequencing in all families that showedpositive linkage to these regions (data not shown). A significantcombined multipoint LOD score of 28.1 was reached in the 3.3 cM intervalflanked by markers D15S778 and D15S659 in the linked kindreds (FIGS. 1and 2). Haplotype reconstructions identified two critical recombinationevents that allowed to restrict the candidate interval to 6.6 cM betweenmarkers D15S1044 and D15S123. The 3.2 cM of the D15S778-D15S659 intervalwas considered to be the region most likely containing the responsiblegene on the basis of homozygosity in all consanguineous patients of twosignificantly linked families; family FSP221 linked to SPG11 with amaximal LOD score of 3.85 and family FSP672 linked to the same locuswith a 2.6 LOD score value (FIGS. 3 and 4). In addition, a genome widescreen performed in family FSP221 at a resolution of 10 cM on allchromosomes only identified three other possible locations withmultipoint lod scores of 2.2 to 2.5 that were excluded using 18additional microsatellite markers (data not shown), therefore highlysupporting linkage to SPG11.

The narrowed interval contained 40 genes in accordance with the NationalCenter for Biotechnology Information (NCBI) and the Ensembl databases.Two were excluded in previous studies (SEMA6D and MAP1A, Stevanin et al,2006). We evaluated 16 additional genes from the interval as candidatesfor SPG11, prioritizing those with a known or putative function inmitochondrial metabolism, intra-cellular trafficking or cytoskeletonintegrity (FIG. 5). All coding and non-coding exons as well as theirsplicing sites with at least 50 bp of intronic sequences on each sidewere sequenced on genomic DNA of 5 index patients from 5 linkedfamilies. No mutations were found in 15 genes but sequence variationswere found in the KIAA1840 gene. We then screened one affected memberfrom the 16 linked families as well as of the uninformative kindreds andchecked all other members of the families, when available, for sequencevariations. 43 different mutations were identified in 47 families,including the 16 linked ones, 31 at the homozygous state, (FIGS. 5 to9). They were either nonsense mutations (n=13), deletions (n=17),insertions (n=7) or splice site mutations (n=6) in the coding sequence,and resulted in an abnormally truncated protein or predicted to alterthe splicing of the messenger RNA in all cases. In two families (FSP670and ITA28 VAC, FIG. 8), we found a missense change (R945G or R815M)affecting a nucleotide of the 5′-splice site consensus and predicted toalter the splicing of the mRNA. This could be confirmed in family FSP670by the analysis of mRNA from one patient (c.2833A>G, r.2834+1_(—)2834+65ins, p.R945GfsX950). Four mutations affected the intronic part of theconsensus sequence for the acceptor splicing site (see Table 2) thatalso likely affect the splicing of the mRNA. The mutations segregatedcompletely in the families with the disease and were not found on atleast 140 chromosomes from unrelated control individuals of European andNorth-African origin suggesting that these mutations were notpolymorphisms. Only 4 mutations were found in more than one pedigree(FIGS. 6 and 7). A c.6100C>T substitution that replaces an arginine by astop codon in exon 32 (R2034X), shortening the protein from 2443 to 2034amino acids (SEQ ID NO:160), was identified, in the homozygous state, in4 Algerian, 3 Tunisian and 2 Moroccan consanguineous kindreds (FIG. 6).A 5 bp deletion in exon 3 (c.529_(—)533delATATT) leading to a frameshiftand a stop codon at aa 178 (SEQ ID NO:150) was found at the homozygousstate in all patients of 3 Portuguese families and at the heterozygousstate in one Brazilian kindred (FIG. 7). Interestingly, alleles at closeflanking markers were partially similar in families with identicalmutations (when it could be tested) suggesting founder effects inNorth-Africa and Portugal for these mutations. The c.733_(—)734 del ATmutation was also found in 4 Tunisian pedigrees, sharing partial commonhaplotypes (data not shown) and one French kindred. Finally, thec.1951C>T variant was found at the heterozygous state in 2 Italian andone Kindred from Romania for which we are extending the pedigrees tocheck for haplotype conservation.

No mutations were found in 44 families, suggesting that the responsiblemutations were either in non-coding regions of KIAA1840 or in anotherunidentified gene.

SPG11 mutations were thus found in the majority of the families with thetypical AR-HSP-TCC studied here (47/91). Most families originated fromthe Mediterranean basin. Complete examination of 22 affected members(Stevanin et al, 2007), 12 men and 10 women, showed a mean age of24.8±9.5 years ranging from 12 to 49. Onset of the disease alwaysoccurred before age 24 years (mean age 11.8±5.5 years; range 2-23) andconsisted in either spastic gait (57%, 12/21) or cognitive impairment(19%, 4/21), sometimes diagnosed as mental retardation. After about 10years of evolution, the full-blown clinical picture consisted inprogressive and severe spastic paraplegia with distal wasting andcognitive problems. In several cases (n=6), cognitive dysfunctionclearly worsened with disease progression. Cerebral imaging showed athin corpus callosum, but also periventricular white matter changes andcortical atrophy, in the majority of the patients. Pseudo-bulbardysarthria was frequent (54%, n=12) and dystonic voice was noted in onepatient. Interestingly, although a few patients had normalelectromyographic recordings, peripheral neuropathy was frequent (72%,13 out of 18 patients) and was mostly associated with pure motorchanges. Additional signs, such as optic atrophy, retinitis pigmentosa,mild cerebellar signs, cataract, and clinodactily were occasionallyobserved, a finding that expands the clinical spectrum of this entity.

The human KIAA1840 gene contains 40 exons spanning 101 Kbases of genomicDNA on chromosome 15q21.1. The full length transcript encodes apredicted protein of 2443 amino acids of unknown function calledspatacsin for SPAsticity with Thin or Atrophied Corpus callosum SyndromeproteIN. The sequence of spatacsin is strongly conserved throughevolution with orthologues in mammalians and other vertebrates: humanKIAA1840 shares 85% identity with the homologous protein in dog, 76 and73% with the mouse and rat homologues and 59% with the chickenhomologue, all of similar sizes. Less similarity was found withhomologous proteins of smaller sizes from fugu (44%), tetraodon (39%),and drosophila (22%).

Neither the gene nor the predicted protein it encodes in many speciesshow any significant sequence similarity to known cDNA or proteinsequences. We then looked for protein motifs and domains (FIG. 3). Fourputative transmembrane domains were predicted by various algorithms (aa163-194, 200-240, 1239-1267 and 1471-1493). A glycosyl hydrolase F1signature was also detected (aa 482-490). This motif is based on aconserved glutamic acid residue which has been shown in thebeta-glucosidase of various bacteria and plants and mammalianlactase-phlorizin hydrolase, an integral membrane glycoprotein thatsplits lactose in the small intestine. Interestingly, this protein isassigned to the aromatic compound dioxygenase superfamily because of a22% identity with the consensus sequence between aa 2104-2381. A leucinezipper (aa 611 to 632), involved in dimerization of many gene-regulatoryproteins (C/EBP, CREB, CRE-BP1, ATFs and Jun/AP1 family of transcriptionfactors) and a Myb domain (aa 1766 to 1774), involved in the DNA-bindingof drosophila and vertebrate myb and related proteins, were alsoidentified. Interestingly, there is a 47% identity, over 44 aa, withthymosyl-like peptides, small peptides which play an important role inthe organization of the cytoskeleton; these peptides, bind to andsequester actin monomers, thereby inhibiting actin polymerization (Lowand Golstein 1985). Furthermore, a probable coiled-coil domain of 33residues from 1556 to 1590 was also present and such domains arereported in structural or motor proteins such as spectrin, laminin,dynein or neurofilament proteins.

We then looked at the structure of the predicted protein. The level ofhydrophobicity (34.2%) over the entire sequence was typical of aglobular protein. Because of it's size, a succession of globular domainsis likely and we tried to identify them by the identification of interdomain regions, corresponding to a low density of hydrophobic clusterswith the DomHCA software (Prat-Albeau et al, 2006). Except a smalllinker located between positions 1410 and 1440, no domain separation wasevidenced. From the HCA plots, one of the putative transmembrane regionswas confirmed at amino acids 200 to 240 on spatacsin from 5 vertebrates,but it was lacking in the homologous sequences from tetraodon anddrosophila, as these last two sequences presented a shortened N-terminaldomain. A thorough analysis of putative duplication highlighted twostructurally similar regions (aa 560-700 and 2250-2390) in allvertebrate homologues of the protein with 19% sequence identity in humansequences (FIG. 10). Amino acid proportion shows a non standarddistribution in some cases: high amount of leucines (13,8% vs 9,6% instandard reference databases), a low level of methionines (1.9% insteadof 2.38%) and glycines (3.9% vs 6.93% in Swiss Prot). The proportion ofcysteins was over 2 fold higher (2,9%) compared to the mean in databasesbut did not gave rise to disulfide bridges, according to the predictionsof the CysState software (Mucchielli-Girgi, 2002). Cluster shapes claimfor a mainly helical behavior of this protein, which is confirmed bystandard prediction tools.

The spatacsin protein, fused with GFP, had a diffuse cytosolic andnuclear distribution that sometimes excluded the nucleus of COS-7 cells.In rare cases (<5%), spatacsin formed small perinuclear dots oraggresome-like structures in cells with high expression levels after 4days post-transfection that did not colocalized with the mitochondrialmarker Cox2 or the Golgi marker alpha-COP.

Previous expression profiling of the SPG11 gene showed that it isexpressed ubiquitously at low levels in mouse tissues, including thebrain (Nagase et al, 2001). Ubiquitous low level expression, even instructures apparently not related to the phenotype, has been shown forother genes responsible for neurodegenerative diseases (Paisan-Ruiz etal, 2004). We successfully amplified seven overlapping cDNA fragmentsfrom the KIAA1840 mRNA extracted from human cerebral cortex and usedthem to probe human adult multiple-tissue northern blots. At least threealternative transcripts were detected in all structures of adult brain.The full-length transcript (˜8 Kb) was most highly expressed in thecerebellum, the 5.5-Kb transcript in the cerebral cortex (FIG. 11).

When the temporal and regional expression of the mouse KIAA1840 mRNA wasinvestigated by in situ hybridization in rat brain, it was undetectablein newborn rats (P1). It was detected in the cerebellum, however, fromP6 to P21. At adulthood (P68), expression was found throughout thebrain. Expression was generally low, but stronger signals were observedin the pineal gland, the edges of the lateral ventricles, the granularlayer of the cerebellum and the hippocampus. In contrast to human adultnorthern blots, only a weak expression was detected in the cerebralcortex. Understanding the function of spatacsin in these structureswould help to explain the major features of the disease phenotype: e.g.,expression in the hippocampus could be related to the cognitiveimpairment observed in the patients. In addition, whether the labelingof the edges of the lateral ventricles, where oligodendrocyteprogenitors are located, is related to the white matter changes inpatients remains to be investigated.

Our study identified the gene responsible for spastic paraplegia withthin corpus callosum linked to SPG11, KIAA1840. This is supported byfour pieces of evidence; first, we have excluded 17 out of the 40 genesassigned to the SPG11 candidate interval; second, we have identified 43different mutations segregating in 47 families, 16 of which linkedpreviously to the SPG11 locus, and not found in at least 140 controlchromosomes; third, all, these mutations were leading to a truncatedprotein and/or abnormally spliced mRNA, and fourth all mutated familiespresented with the typical AR-HSP-TCC phenotype or at least a compatiblephenotype in 2 families in which cerebral imaging was not available.Mutations in KIAA1840 affected 47 of 91 AR-HSP-TCC families in thisstudy making this genetic entity very frequent among AR-HSP-TCC (52%),75% was estimated in a previous study (Stevanin et al, 2006), but alsoamong recessive spastic paraplegias. At least another gene might howeverexist as previously shown (Lossos et al, 2006; Stevanin et al, 2006;Casali et al, 2004).

This gene has a widespread low level expression, including in the brainwhere it is more strongly expressed in the cerebellum, the cerebralcortex, the hippocampus, the pineal gland and the edges of theventricles. Spastic paraplegias are supposed to results from a dyingback mechanism of the exons and mitochondrial metabolism or axonaltransport has been implicated in several genetic entities of HSP (Crosbyet al, 2002). Indeed, three causative genes identified in AR-HSP havebeen implicated in defective intracellular trafficking: mutations in themitochondrial metalloprotease protein paraplegin impair axonal transportin SPG7; spartin (SPG20) mutations affect endosomal trafficking andmicrotubule dynamics; maspardin (SPG21) mutations may interfere withendosomal/trans-Golgi vesicle transportation. Although, the function ofspatacsin remains unknown, given it's basal expression in all tissuesand it's high conservation in all species, this protein might have acrucial function which might explain the degeneration of thecorticospinal tracts which might rely on the post-translationalmodifications or modeling/carriage of other proteins involved in axonaltransport, mitochondrial metabolism as well as cerebral development. Thepresence of at least one transmembrane domain suggests that spatacsinmay act as a receptor of a transporter.

All mutations identified so far in the KIAA1840 gene cause or arepredicted to cause truncation of the protein, suggesting thatpathogenicity results from loss of function. They are located in manyexons, including exon 1 and exon 39 suggesting that the C-terminaldomain of the protein has also an important function or effect on thestructure of the protein. It is also conceivable that, given itsposition in the 5′splice site consensus sequence, the missense mutationR815M would also affect the transcription of the gene as demonstratedfor mutation c.2833A>G, r.2834+1_(—)2834+65 ins, p.R945GfsX950.Similarly, the mutations found in the intronic part of the acceptorsplicing sites in introns 4, 12, 13 and 34 (Table 2) are likely alteringthe splicing of the surrounding exons and therefore the synthesis and/orstability of the mRNA or protein. No tissues from patients wereavailable yet, however, to validate this hypothesis.

The identification of the SPG11 gene will now improve the diagnosticprocedure, as well as patient management, and permit more accurategenetic counseling. This is invaluable for patients and their families.

REFERENCES

The following are all incorporated herein by reference:

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1-17. (canceled)
 18. A method for detecting the presence of a KIAA1840nucleic acid mutation in a subject with a family history of hereditaryspastic paraplegia (HSP), which method comprises: assaying a samplecontaining a KIAA1840 nucleic acid from said subject to determine thepresence of a mutated KIAA1840 nucleic acid as compared to a controlpopulation, wherein said mutated KIAA1840 nucleic acid sequence is atleast 90% identical to SEQ ID NO:1 and encodes a truncated form of theKIAA protein, and detecting the mutation in the sample.
 19. The methodof claim 18, wherein the KIAA 1840 mutation is a substitution selectedfrom the group consisting of c.6100C>T, c.2198T>G, c.118C>T, c.1235C>G,c.2833A>G, c.1951C>T, c.869+1G>A, c. 1679 C>G, c.2316+1G>A, c.2444G>T,c.2444+1G>C, c.2697G>A, c.5470C>T, c.5870C>G, c.6091C>T, c.6477+4 A>G,c.6856C>T, c.1282A>T, and c.5974C>T.
 20. The method of claim 18, whereinthe KIAA 1840 mutation is a deletion selected from the group consistingof c.529-533delATATT, c.6451delG, c.6832_(—)6833delAG, c.1203delA,c.1549_(—)1550delCT, c.6737_(—)6740delTTGA, c.1471_(—)1472delCT,c.1692delA, c.2716delC, c.1668delT, c.704_(—)705delAT,c.5989_(—)5992delCTGT, c.5532_(—)5533delCA, c.5769delT,c.6739_(—)6742delGAGT, c.4307_(—)4308delAA and c.733_(—)734delAT. 21.The method of claim 18, wherein the KIAA 1840 mutation is an insertionselected from the group consisting of c.7029_(—)7030insT,c.2850_(—)2851insT, c.3741_(—)3742insA, c.5982_(—)5983insCTCT,c.5986_(—)5987insT, c.3075_(—)3076insA and c.2842_(—)2843insG.
 22. Themethod of claim 18, wherein the encoded truncated KIAA1840 protein hasan amino acid sequence selected from the group consisting of SEQ ID NO:149 to SEQ ID NO:164 and SEQ ID NO:166 to SEQ ID NO:188.
 23. The methodof claim 18 wherein said sample is obtained from a source selected fromthe group consisting of blood, blood cells, plasma, serum, lymph, buccalcells, epithelial cells, fibroblasts, and cells present in a tissueobtained by biopsy.
 24. The method of claim 23, wherein said sample isan DNA sample.
 25. The method of claim 24, wherein the sample comprisesgenomic DNA and the nucleic acid in the sample is amplified usingoligonucleotide primers that are specific for a mutated site in the KIAA1840 nucleic acid or that are capable of amplifying a region containingthe mutation.
 26. The method of claim 25, wherein the method furthercomprises DNA sequencing.
 27. The method of claim 23 wherein said sampleis an RNA sample.
 28. The method of claim 27, wherein the sample issubjected to coupled reverse transcription and nucleic acidamplification using oligonucleotide primers specific for a mutated sitein the KIAA 1840 nucleic acid or amplify a region containing themutation.
 29. The method of claim 28, wherein the method furthercomprises DNA sequencing.
 30. The method of claim 18, wherein the sampleis assayed by at least one process selected from the group consisting ofdirect sequencing, restriction fragment length polymorphism (RFLP)analysis, hybridization with allele-specific oligonucleotides,allele-specific PCR, PCR using mutagenic primers, ligase-PCR, HOTcleavage, denaturing gradient gel electrophoresis, temperaturedenaturing gradient gel electrophoresis, single stranded conformationalpolymorphism and denaturing high performance liquid chromatography. 31.An ex vivo method of diagnosing or predicting a hereditary spasticparaplegia (HSP) in a subject with a family history of hereditaryspastic paraplegia (HSP), which method comprises: assaying a samplecontaining a KIAA1840 nucleic acid from said subject to determine thepresence of a mutated KIAA1840 nucleic acid as compared to a controlpopulation, wherein said mutated KIAA1840 nucleic acid sequence is atleast 90% identical to SEQ ID NO:1 and encodes a truncated form of theKIAA protein, and detecting the mutation in the sample, therebyindicating the subject has, or is likely to develop, HSP.
 32. The methodof claim 31, wherein the KIAA 1840 mutation is a substitution selectedfrom the group consisting of c.6100C>T, c.2198T>G, c.118C>T, c.1235C>G,c.2833A>G, c.1951C>T, c.869+1G>A, c. 1679 C>G, c.2316+1G>A, c.2444G>T,c.2444+1G>C, c.2697G>A, c.5470C>T, c.5870C>G, c.6091C>T, c.6477+4 A>G,c.6856C>T, c.1282A>T, and c.5974C>T.
 33. The method of claim 31, whereinthe KIAA 1840 mutation is a deletion selected from the group consistingof c.529-533delATATT, c.6451delG, c.6832_(—)6833delAG, c.1203delA,c.1549_(—)1550delCT, c.6737_(—)6740delTTGA, c.1471_(—)1472delCT,c.1692delA, c.2716delC, c.1668delT, c.704_(—)705delAT,c.5989_(—)5992delCTGT, c.5532_(—)5533delCA, c.5769delT,c.6739_(—)6742delGAGT, c.4307_(—)4308delAA and c.733_(—)734delAT. 34.The method of claim 31, wherein the KIAA 1840 mutation is an insertionselected from the group consisting of c.7029_(—)7030insT,c.2850_(—)2851insT, c.3741_(—)3742insA, c.5982_(—)5983insCTCT,c.5986_(—)5987insT, c.3075_(—)3076insA and c.2842_(—)2843insG.
 35. Themethod of claim 31, wherein the encoded truncated KIAA1840 protein hasan amino acid sequence selected from the group consisting of SEQ ID NO:149 to SEQ ID NO:164 and SEQ ID NO:166 to SEQ ID NO:188
 36. The methodof claim 31, wherein nucleic acid in the sample is amplified usingoligonucleotide primers capable of amplifying a region containing theKIAA1840 mutation.
 37. The method of claim 36 further comprising DNAsequencing.